Compositions and methods for quadricyclane modification of biomolecules

ABSTRACT

The present disclosure features a strain-promoted [2+2+2] reaction that can be carried out under physiological conditions. In general, the reaction involves reacting a pi-electrophile with a low lying LUMO with a quadricyclane on a biomolecule, generating a covalently modified biomolecule. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo and in vitro. The reaction is compatible with modification of living cells. In certain embodiments, the pi-electrophile can comprise a molecule of interest that is desired for delivery to a quadricyclane-containing biomolecule via [2+2+2] reaction.

CROSS-REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 13/605,695, filed Sep. 6, 2012, now issued as U.S. Pat. No.9,556,195, which claims the benefit of U.S. Provisional PatentApplication No. 61/533,607, filed Sep. 12, 2011, which applications areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM058867awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Bioorthogonal transformations include four broad reaction types:1,3-dipolar cycloadditions, Diels-Alder reactions, metal-catalyzedcouplings, and nucleophilic additions. Outside of this space lies the[2+2+2] cycloaddition reaction, a choice for the rapid assembly offunctionalized ring systems. In practice, such reactions are typicallymetal catalyzed as a means to overcome an otherwise significant entropicbarrier. However, the highly strained hydrocarbon quadricyclane directlyundergoes [2+2+2] cycloaddition with specific types of pi systems.

LITERATURE

U.S. Pat. No. 7,808,619; U.S. Patent Publication No. 2009/0068738; U.S.Pat. No. 6,570,040

SUMMARY

The present disclosure features a strain-promoted [2+2+2] reaction thatcan be carried out under physiological conditions. In general, thereaction involves reacting a pi-electrophile with a low lying LUMO witha quadricyclane on a biomolecule, generating a covalently modifiedbiomolecule. The selectivity of the reaction and its compatibility withaqueous environments provides for its application in vivo and in vitro.The reaction is compatible with modification of living cells. In certainembodiments, the pi-electrophile can comprise a molecule of interestthat is desired for delivery to a quadricyclane-containing biomoleculevia [2+2+2] reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a spectrum that shows that quadricyclane is stable inphosphate buffered saline (PBS, pH 7.4). 7-acetoxy quadricyclane (1, 2mg) was dissolved in 0.4 mL of CD₃CN. To this solution, 0.4 mL ofdeuterated PBS was added. The NMR spectrum above was taken 2.5 monthsafter the described solution was prepared.

FIG. 2 is a spectrum that shows that quadricyclane is stable tocysteine. 7-acetoxy quadricyclane (1, 2 mg) was dissolved in 0.4 mL ofCD₃CN. To this solution, 0.4 mL of deuterated PBS and 3 mg of cysteinewere added. The NMR spectrum above was taken 2.5 months after thedescribed solution was prepared.

FIG. 3 is a spectrum that shows that Complex 3 is photo-labile. Complex3 (2.2 mg) was dissolved in CDCl₃ (˜0.75 mL) and placed in an NMR tubeon the bench continually exposed to ambient light. NMR spectra wereperiodically obtained. The asterisks indicate the chemical shifts for7-acetoxy norbornadiene (4). At the 36 h time point, there is a 1:1.1ratio of 3:4 as judged by integration of the olefin peaks. Thecalculated half-life based on all integration data is 34.8 h.

FIG. 4 is a spectrum that shows that Complex 3 is photo-labile. Using anNMR assay degradation of 3 over the first few hours is not evident.However, using UV/Vis/NIR spectroscopy, which has greater sensitivitythan NMR, the formation of 2 from 3 is evident at early timepoints. Asolution of 3 was prepared in CH₃CN and left in ambient light. AUV/Vis/NIR spectrum was obtained every 15 min. The NIR absorption bandat 855 nm characteristic of 2 is growing in, while the small humpcentered at ˜400 nm characteristic of the product is decreasing. Theabsorption band at 350 nm is also increasing in intensity.

FIGS. 5A and 5B are spectra that show that diethyldithiocarbamate (5)prevents the photo-degradation of complex 3. A solution containing 200μM 3 and 0 (red) or 1.25 (green) mM 5 in 3:1 CH₃CN/H₂O was prepared andleft in ambient light continually. A. UV/Vis/NIR spectra of thedescribed solutions taken before being exposed to light. B. UV/Vis/NIRspectra of the described solutions taken after 20 h of exposure tolight. The red line contains little absorbance due to the low solubilityof 2 in acetonitrile. If 2 is reduced to the anionic state by cysteine(1.25 mM, blue line) solubility in acetonitrile is improved and evidenceof photodegradation can be seen. The diethyldithiocarbamate treatedsample remains unaltered after exposure to light indicating nophotodegradation occurred.

FIG. 6 is a spectrum that monitors the reaction between 1 and 14. Aseries of UV/Vis/NIR spectra taken as the reaction in part A isproceeding. A solution of 14 (400 μM in PBS) was combined with 1 (20 mMin EtOH) and a UV/Vis/NIR spectrum was taken every 30 seconds.

FIGS. 7A and 7B are graphs that show the second-order rate constant forthe reaction of 1 and 14 was determined using pseudo-first orderkinetics. A solution of 400 μM 14 in PBS was combined with varioussolutions of quadricyclane 1 (20 mM, 16 mM, 12 mM, 8 mM, 4 mM, or 0 mMin EtOH) in a 1:1 ratio (total volume=100 μL). The reaction wasmonitored by the absorbance at 850 nm for 15 min. The absorbance valueswere correlated to the concentration of 14 using a standard curve. Aplot of Ln[14] verses time resulted in a series of first-order rateconstants (k_(obs)). Plotting each k_(obs) value vs. [1] yields a linearregression with the slope of the line indicating the second-order rateconstant. The average of nine trials resulted in a second-order rateconstant of 0.25±0.05 M⁻¹ s⁻¹. A. A representative plot to determinek_(obs). B. Plot of k_(obs) vs. [1] for each trial.

FIG. 8 is a graph that shows the stability of Ni bis(dithiolene) 14 isstable to PBS. Ni bis(dithiolene) 14 was dissolved in PBS. Theabsorption of the NIR band was monitored for changes over 20 h. AUV/Vis/NIR spectrum was taken every hour. Only slight reduction insignal is evident.

FIGS. 9A-9C are graphs showing the stability of Ni bis(dithiolene) 14 ismoderately stable to excess of free amino acids. A solution of 400 μM 14in PBS was combined with solutions of 50 mM of the indicated amino acidin a 1:1 ratio. The absorbance of each solution was monitored over time.A. The UV/Vis/NIR spectra taken after approximately 15 min. B. TheUV/Vis/NIR spectra taken after 1 h. C. The UV/Vis/NIR spectra takenafter 2 h. Red=no amino acid present. Green=arginine. Blue=histidine.Black=glycine. Purple=serine.

FIGS. 10A and 10B show that Ni bis(dithiolene) 14 is not stable tocysteine. A. Schematic for the reduction of 14 to 12 by free cysteine.B. UV/Vis/NIR analysis of the reaction between 14 and cysteine. Asolution of 125 μM 14 in PBS was prepared with 0 (red), 1 (blue), 10(green) or 100 (black) equivalents of cysteine. All the mixturesinstantly turned orange upon addition of cysteine and displayed theUV/Vis/NIR spectra shown above. A smaller, red-shifted NIR absorptionband is consistent with reduction of 14 to 12.

FIGS. 11A-11C are graphs showing that Ni bis(dithiolene) 14 is notstable to reducing agents. A solution of 125 μM 14 in PBS was preparedwith 0 (red), 1 (blue, solid), 10 (green, solid) or 100 (black, solid)equivalents of β-mercaptoethanol (BME, A), tris(carboxyethyl)phosphine(TCEP, B), or N-acetyl cysteine (C). All the mixtures instantly turnedorange upon addition of reducing agent and displayed the UV/Vis/NIRspectra shown above. A smaller, red-shifted NIR absorption band isconsistent with reduction of 14 to 12. For the cases of 1 and 10equivalents of reducing agent, 14 could be recovered by the addition of3 equivalents or 30 equivalents of potassium ferrocyanide (K₃Fe(CN)₆),respectively (blue or green dashed lines). However, the addition of 300equivalents of K₃Fe(CN)₆ did not restore 14 when 14 was subjected to 100equivalents of reducing agent (black dashed line).

FIGS. 12A and 12B show that Ni bis(dithiolene) 14 can be rescued byaddition of K₃Fe(CN)₆. A. Schematic for the oxidation of 12 to 14. B.UV/Vis/NIR analysis of the oxidation. A solution of 125 μM 14 in PBS wastreated with 1, 10, or 100 equivalents of cysteine followed by 3, 30, or300 equivalents of K₃Fe(CN)₆, respectively (blue, green, black dashedlines). After 1 h, UV/Vis/NIR spectra were collected. The red linerepresents 125 μM 14 with no treatment.

FIG. 13 is a graph that shows that Ni bis(dithiolene) 14 rescued by theaddition of K₃Fe(CN)₆ reacts with quadricyclane. A solution of 125 μM 14in PBS was treated with 1 equivalent of cysteine followed by 3equivalents of K₃Fe(CN)₆. Quadricyclane was added and a UV/Vis/NIRspectrum was immediately recorded (red line). UV/Vis/NIR spectra werethen collected every 1 min and 40 sec. The observed reduction in signalis consistent with the quadricyclane ligation occurring (See Figure S6).

FIGS. 14A and 14B are graphs showing that Ni bis(dithiolene) 14 isreduced by bovine serum albumin (BSA). A solution containing 14 (100 μM)and BSA (1.2 equivalents (A) or 12 equivalents (B)) was monitored byUV/Vis/NIR spectroscopy at various timepoints over 20 h. UV/Vis/NIRspectra were normalized to each other at 650 nm.

FIG. 15 is a graph that shows that Ni bis(dithiolene) 14 is stable tooxidized insulin over multiple hours. A solution containing 14 (100 μM)and oxidized insulin (1.2 equivalents) was monitored by UV/Vis/NIRspectroscopy at various timepoints over 20 h. UV/Vis/NIR spectra werenormalized to each other at 650 nm.

FIGS. 16A-16E show that Ni bis(dithiolene) reagents selectively labelquadricyclane-modified BSA. A. Modification of BSA with quadricyclaneand subsequent labeling with a biotinylated nickel dithiolene reagent.B/C. Ni bis(dithiolene) 15 displays time (B)- and dose (C)-dependentlabeling of QC-BSA. BSA (−) or QC-BSA (+) (5 μg) was treated with 50 μM15 for various amounts of time (B) or various concentrations of 15 for30 min (C). The reactions were quenched with 1 and 5 and the presence ofproduct was detected by Western blot using α-biotin-HRP. D. Reaction of15 with QC-BSA can be prevented by pretreatment with tetrasulfonated Nibis(dithiolene) 17. BSA (−) or QC-BSA (+) (5 μg) was treated withvarying amounts of 17 for 30 min followed by 50 μM of 15 for 30 min. Thereactions were quenched with 1 and 5 and the samples were analyzed byWestern blot probing with α-biotin-HRP. E. QC-BSA can be selectivelylabeled in a mixture of proteins. To 25 μg of lysate in the presence of1 mM K₃Fe(CN)₆ was added no BSA or reagent (lane 1), BSA (1.5 μg) and 50μM 15 (lane 2), or QC-BSA (1.5 μg) and 50 μM 15 (lane 3). After 30 min,the reactions were quenched with 1 and 5 and analyzed by Western blot.The BSA monomer and dimer bands are visible in the QC-BSA treatedsample. The bands denoted with an asterisk represent an endogenouslybiotinylated E. coli protein. B-E. Equal protein loading was verified byPonceau stain.

FIG. 17 shows that Oxidizing agent increases the efficiency of labelingof QC-BSA by 15. BSA (1.5 μg, lanes 1,3) or QC-BSA (1.5 μg, lanes 2,4)were combined with 15 (50 μM) for 30 min in the presence (lanes 3-4) orabsence (lanes 1-2) of 1 mM K₃Fe(CN)₆. Lysate from E. coli (25 μg) wascombined with (lane 6) or without (lane 5) 1 mM K₃Fe(CN)₆ for 30 min.Lysate (25 μg) and BSA (1.5 μg, lanes 7,9) or QC-BSA (1.5 μg, lanes8,10) were combined with 15 (50 μM) for 30 min in the presence (lanes9,10) or absence (lanes 7,8) of 1 mM K₃Fe(CN)₆. After 30 min, allreaction mixtures were quenched with excess 1 and 5 and analyzed byWestern blot probing with α-biotin-HRP. Protein loading was verified byPonceau Stain. The bands denoted with an asterisk represent anendogenously biotinylated E. coli protein.

FIGS. 18A and 18B show that the quadricyclane ligation is orthogonal toCu-free click chemistry and the oxime ligation. A. A mixture of 8 μg ofQC-BSA, AzDHFR, and CHO-MBP was treated with 15 (150 μM), DIMAC-fluor(250 μM), and H₂NO-FLAG (1 mM) for 3 h at 37° C., pH 4.5. This mixturewas basified with 850 mM tris buffer and quenched with excess 1, 5, and2-azidoethanol. It was then separated into 3 portions and each portionwas analyzed by Western blot probing with a different antibody:α-biotin-HRP (quadricyclane ligation), α-fluorescein-HRP (Cu-free clickchemistry) or α-FLAG-HRP (oxime ligation). The Ponceau stain indicatesall three proteins were present. Oligomer bands are observed for BSA andDHFR.

FIGS. 19A-19F show controls for the banding patterns seen in FIG. 18B.A/B. QC-BSA (8 μg) and 15 (150 μM) were combined 37° C., pH 4.5. After 3h, this mixture was basified with 850 mM tris buffer and quenched withexcess 1, 5, and 2-azidoethanol. It was then analyzed by Western blotprobing with an α-biotin-HRP (B). C/D. CHO-MBP (8 μg) and H₂NO-FLAG (1mM) were combined at 37° C., pH 4.5. After 3 h, this mixture wasbasified with 850 mM tris buffer and quenched with excess 1, 5, and2-azidoethanol. It was then analyzed by Western blot probing with anα-FLAG-HRP antibody (D). E/F. AzDHFR (8 μg) and DIMAC-fluor (250 μM)were combined at 37° C., pH 4.5. After 3 h, this mixture was basifiedwith 850 mM tris buffer and quenched with excess 1, 5, and2-azidoethanol. It was analyzed by Western blot probing with anα-fluorescein-HRP antibody (F).

FIG. 20 shows cytotoxicity of 17 and the adduct of 1 and 17 in relationto NiCl₂ and Cu(I). Jurkat cells were washed twice with FACS buffer (PBSwith 1% FBS) and placed in a 96-well plate with ˜400,000 cells/well(pellet 2500×g, 3 min, 4° C.). The cells were treated with at 0, 10, 25,50, 100, 250, or 500 μM of 17 (blue diamond), the product of 1 and 17(red square), NiCl₂ (purple triangle), or CuSO₄ in the presence of 1 mMTCEP (green cross) for 1 h. The cells were washed three times byresuspension in FACS buffer (200 μL) followed by concentration bycentrifugation (2500×g, 3 min, 4° C.). Following the third wash, thecells were resuspended in 100 μL of 1× binding buffer containing 5 μL of7-AAD and 5 μL of FITC-AnnexinV (buffer and reagents from BDPharmingen™). The cells were incubated at rt in the dark for 15 min,diluted to 500 μL with binding buffer and analyzed by flow cytometry(FL1 vs. FL3) on a BD Biosciences FACSCalibur flow cytometer equippedwith a 488-nm argon laser. Plotted is the percentage of cells that donot stain with either 7-AAD or FITC-Annexin-V. The error bars representthe standard deviation of three replicate samples.

FIGS. 21A-21C show representative dot plots for the experiment in FIG.20. A. Cells treated with no reagent. B. Cells treated with 50 μM ofreagent. C. Cells treated with 500 μM reagent. The percentage of cellsin the bottom left quadrant is what is plotted in FIG. 20. MFI=meanfluorescence intensity (arbitrary units).

FIG. 22 shows cytotoxicity of diethyldithiocarbamate (5). Jurkat cellswere washed twice with FACS buffer (PBS with 1% FBS) and placed in a96-well plate with ˜500,000 cells/well (pellet 2500×g, 3 min, 4° C.).The cells were treated with 0, 1.25, 2.5, or 5.0 mM of 5 for 1 h. Thecells were washed three times by resuspension in FACS buffer (200 μL)followed by concentration by centrifugation (2500×g, 3 min, 4° C.).Following the third wash, the cells were resuspended in 100 μL of 1×binding buffer and 7.5 μL of 7-AAD and 5 μL of AnnexinV-PE were added(buffer and reagents from BD Pharmingen™). The cells were incubated atrt in the dark for 15 min, diluted to 500 μL with binding buffer andanalyzed by flow cytometry (FL2 vs. FL3) on a BD Biosciences FACSCaliburflow cytometer equipped with a 488-nm argon laser. Plotted is thepercentage of cells that do not stain with either 7-AAD or AnnexinV-PE.The error bars represent the standard deviation of three replicatesamples.

FIG. 23 shows representative dot plots for the experiment in Figure S20.The percentage of cells in the bottom left quadrant is what is plottedin Figure S20. MFI=mean fluorescence intensity (arbitrary units).

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

By “reaction partner” is meant a molecule or molecular moiety thatspecifically reacts with another reaction partner. Exemplary reactionpartners are those of a subject reaction, i.e., a quadricyclane group ofa quadricyclane-modified biomolecule and a pi-electrophile comprising amolecule of interest.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment or its syntheticenvironment and is at least 60% free, at least 75% free, at least 90%free, at least 95% free, at least 98% free, or at least 99% free fromother components with which it is naturally associated, or is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, or at least 99% free from contaminants associated withsynthesis of the compound.

As used herein, the term “cell” in the context of in vivo and ex vivoapplications is meant to encompass eukaryotic and prokaryotic cells ofany genus or species, e.g., mammalian cells. “Cell” is also meant toencompass both normal cells and diseased cells, e.g., cancerous cells.In many embodiments, the cells are living cells.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups —NR²⁰C(O)alkyl, —NR²⁰C(O)substitutedalkyl, NR²⁰C(O)cycloalkyl, —NR²⁰C(O)substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O)substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic, provided that the point of attachment is through anatom of the aromatic aryl group. This term includes, by way of example,phenyl and naphthyl. Unless otherwise constrained by the definition forthe aryl substituent, such aryl groups can optionally be substitutedwith from 1 to 5 substituents, or from 1 to 3 substituents, selectedfrom acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, substituted cycloalkyl,substituted cycloalkenyl, amino, substituted amino, aminoacyl,acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl,cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substitutedthioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substitutedalkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups—O—C(O)O-alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl,—O—C(O)O-substituted alkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substitutedalkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl,—O—C(O)O-substituted cycloalkenyl, —O—C(O)O-heteroaryl,—O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclic, and—O—C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen, and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl, imidazolyl or furyl) or multiple condensedrings (e.g., indolizinyl, quinolinyl, benzimidazolyl or benzothienyl),wherein the condensed rings may or may not be aromatic and/or contain aheteroatom, provided that the point of attachment is through an atom ofthe aromatic heteroaryl group. In certain embodiments, the nitrogenand/or sulfur ring atom(s) of the heteroaryl group are optionallyoxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonylmoieties. This term includes, by way of example, pyridinyl, pyrrolyl,indolyl, thiophenyl, and furanyl. Unless otherwise constrained by thedefinition for the heteroaryl substituent, such heteroaryl groups can beoptionally substituted with 1 to 5 substituents, or from 1 to 3substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl, andtrihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 15 ring atoms, including 1 to 4 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂-moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group SO₂-alkyl, SO₂-substituted alkyl,SO₂-alkenyl, SO₂-substituted alkenyl, SO₂-cycloalkyl, SO₂-substitutedcycloalkyl, SO₂-cycloalkenyl, SO₂-substituted cylcoalkenyl, SO₂-aryl,SO₂-substituted aryl, SO₂-heteroaryl, SO₂-substituted heteroaryl,SO₂-heterocyclic, and SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein. Sulfonyl includes, by way of example, methyl-SO₂—, phenyl-SO₂—,and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, OSO₂-substituted alkyl,OSO₂-alkenyl, OSO₂-substituted alkenyl, OSO₂-cycloalkyl,OSO₂-substituted cycloalkyl, OSO₂-cycloalkenyl, OSO₂-substitutedcylcoalkenyl, OSO₂-aryl, OSO₂-substituted aryl, OSO₂-heteroaryl,OSO₂-substituted heteroaryl, OSO₂-heterocyclic, and OSO₂ substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N—OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸⁰, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰;—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ isselected from the group consisting of optionally substituted alkyl,cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each R⁷⁰ is independentlyhydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, twoR⁸⁰'s, taken together with the nitrogen atom to which they are bonded,form a 5-, 6- or 7-membered heterocycloalkyl which may optionallyinclude from 1 to 4 of the same or different additional heteroatomsselected from the group consisting of O, N and S, of which N may have —Hor C₁-C₃ alkyl substitution; and each M⁺ is a counter ion with a netsingle positive charge. Each M⁺ may independently be, for example, analkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; oran alkaline earth ion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or[Ba²⁺]_(0.5) (“subscript 0.5 means e.g. that one of the counter ions forsuch divalent alkali earth ions can be an ionized form of a subjectcompound and the other a typical counter ion such as chloride, or twoionized compounds of the present disclosure can serve as counter ionsfor such divalent alkali earth ions, or a doubly ionized subjectcompound can serve as the counter ion for such divalent alkali earthions). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl andN-morpholinyl.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on unsaturated carbon atoms in“substituted” alkene, alkyne, aryl and heteroaryl groups are, unlessotherwise specified, —R⁶⁰, halo, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰,trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, —N₃, —SO₂R⁷⁰, —SO₃ ⁻M⁺,—SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃ ⁻M⁺, —OSO₃R⁷⁰, —PO₃ ⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —CO₂ ⁻M⁺, —CO₂R⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰, —OCO₂⁻M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺,—NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined, provided that in case of substituted alkene or alkyne, thesubstituents are not —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, or —S⁻M⁺.

In addition to the disclosure herein, substituent groups for hydrogenson nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkylgroups are, unless otherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰,—S⁻M⁺, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰,—S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰, —OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰,—P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰,—C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰,—NR⁷⁰C(NR⁷⁰)R⁷⁰, and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ areas previously defined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups are limited to substituted aryl-(substitutedaryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. “Pharmaceutically acceptable salt” refers to pharmaceuticallyacceptable salts of a compound, which salts are derived from a varietyof organic and inorganic counter ions well known in the art and include,by way of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, formate, tartrate, besylate, mesylate,acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Where applicable, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aquadricyclane” includes a plurality of such quadricyclanes and referenceto “the biomolecule” includes reference to one or more biomolecules andequivalents thereof known to those skilled in the art, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Representative Embodiments

The present disclosure features a strain-promoted [2+2+2] reaction thatcan be carried out under physiological conditions. In general, thereaction involves reacting a pi-electrophile with a low lying lowestunoccupied molecular orbital (LUMO) with a quadricyclane on abiomolecule, generating a covalently modified biomolecule. Theselectivity of the reaction and its compatibility with aqueousenvironments provides for its application in vivo and in vitro. Thereaction is compatible with modification of living cells. In certainembodiments, the pi-electrophile can comprise a molecule of interestthat is desired for delivery to a quadricyclane-containing biomolecule(e.g., a biomolecule present in a living cell) via [2+2+2] reaction.

The disclosure provides methods and compositions for specifically andefficiently synthetically modifying cellular components in an aqueousenvironment, thus providing for modification of such cellular componentson or in living cells. The embodiments use reaction partners that arecompletely abiotic and are chemically orthogonal to native cellularcomponents, thus providing for extreme selectivity of the reaction.Furthermore, the reaction can be carried out under physiologicalconditions, e.g., a pH of about 7 within an aqueous environment, and atabout 37° C.

Quadricyclane possesses many qualities that render it a promisingbioorthogonal reagent. First, it is abiotic, and quadricyclane's allsp³-hybridized carbon system would be unreactive with nativebiomolecules. Second, the molecule is relatively small and can thereforebe amenable to biosynthetic incorporation into biomolecules. Also, thestructure of quadricyclane has about 80 kcal/mol of strain that promotes[2+2+2] cycloaddition with pi-systems with a low lying LUMO under mildconditions. The rates of these cycloaddition reactions are greatlyenhanced by the “on water” effect, e.g., the reaction rate is enhancedin aqueous environments.

Quadricyclane reacts with a variety of electrophilic reagents. The highstrain energy of quadricyclane activates the sigma bonds within thismolecule for [2+2+2] cycloaddition with electrophilic reagents.

The reaction partners for the strain-promoted [2+2+2] reaction arediscussed in more detail below. In the description, reference to formulawith a Roman numeral, such as (I), is meant to include the formulae withthe Roman numeral and letter, e.g. (Ia) and (Ib).

Pi-Electrophile Reaction Partner

As discussed above, quadricyclane reacts with a variety of electrophilicreagents. The ability of quadricyclane to react with electrophilicreagents can be attributed to the high strain energy of quadricyclane.In certain embodiments, the electrophilic reagent is a pi-electrophilewith a low lying LUMO. An example of pi-electrophile with a low lyingLUMO is an electron deficient pi-electrophile. Electron deficientpi-electrophile can be attributed to electronegative substituents.Certain pi-electrophile reaction partners (pi-electrophile compounds),including metal bis(dithiolene compounds, azo compounds, alkynylcompounds, alkenyl compounds, and aryl compounds, are discussed below.

Metal Bis(Dithiolene) Compounds

The embodiments provide metal bis(dithiolene) compounds; andcompositions comprising the compounds. A subject metal bis(dithiolene)compound is a compound of the formula:

-   -   wherein    -   M is selected from one of the following: nickel (II), palladium        (II), platinum (II), cobalt (I), iridium (I), rhodium (I),        copper (II), copper (III), silver (III), gold (III), tungsten,        and iron;    -   Ar¹, Ar², Ar³, and Ar⁴ are optional and are aryl, substituted        aryl, heteroaryl, or substituted heteroaryl groups;    -   R¹, R², R³, and R⁴ are optional and are independently selected        from hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹, Y², Y³, and Y⁴ are independently selected from hydrogen;        halogen; a moiety that comprises a reactive group that        facilitates covalent attachment of a molecule of interest; and a        molecule of interest;    -   wherein at least one of Y¹, Y², Y³, and Y⁴ is a moiety that        comprises a reactive group that facilitates covalent attachment        of a molecule of interest or a molecule of interest.

The embodiments provide metal bis(dithiolene) compounds; andcompositions comprising the compounds. A subject metal bis(dithiolene)compound is a compound of the formula:

-   -   wherein    -   M is selected from one of the following: nickel (II), palladium        (II), platinum (II), cobalt (I), iridium (I), rhodium (I),        copper (II), copper (III), silver (III), gold (III), tungsten,        and iron;    -   R¹, R², R³, and R⁴ are optional and are independently selected        from hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹, Y², Y³, and Y⁴ are independently selected from hydrogen;        halogen; a moiety that comprises a reactive group that        facilitates covalent attachment of a molecule of interest; and a        molecule of interest;    -   wherein at least one of Y¹, Y², Y³, and Y⁴ is a moiety that        comprises a reactive group that facilitates covalent attachment        of a molecule of interest or a molecule of interest.

The embodiments provide metal bis(dithiolene) compounds; andcompositions comprising the compounds. A subject metal bis(dithiolene)compound is a compound of the formula:

-   -   wherein    -   M is selected from one of the following: nickel (II), palladium        (II), platinum (II), cobalt (I), iridium (I), rhodium (I),        copper (II), copper (III), silver (III), gold (III), tungsten,        and iron;    -   R² and R³ are independently selected from the following:        hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy;    -   R¹ and R⁴ are optional and are independently selected from        hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹ and Y⁴ are independently selected from hydrogen; halogen; a        moiety that comprises a reactive group that facilitates covalent        attachment of a molecule of interest; and a molecule of        interest;    -   wherein at least one of Y¹ and Y⁴ is a moiety that comprises a        reactive group that facilitates covalent attachment of a        molecule of interest or a molecule of interest.

In formula (I), M is selected from one of the following: nickel (II),palladium (II), platinum (II), cobalt (I), iridium (I), rhodium (I),copper (II), copper (III), silver (III), gold (III), tungsten, and iron.In certain embodiments, M is selected from one of the following: nickel(II), palladium (II), and platinum (II). In certain embodiments, M isnickel (II). In certain embodiments, M is palladium (II). In certainembodiments, M is platinum (II). In certain embodiments, M is selectedfrom one of the following: gold (III), tungsten, and iron. In certainembodiments, M is gold (III). In certain embodiments, M is tungsten. Incertain embodiments, M is iron.

In formula (I), Ar¹, Ar², Ar³, and Ar⁴ are optional and are aryl,substituted aryl, heteroaryl, or substituted heteroaryl groups. Incertain embodiments, Ar¹, Ar², Ar³, or Ar⁴ is aryl. In certainembodiments, Ar¹, Ar², Ar³, or Ar⁴ is substituted aryl. In certainembodiments, Ar¹, Ar², Ar³, or Ar⁴ is heteroaryl. In certainembodiments, Ar¹, Ar², Ar³, or Ar⁴ is substituted heteroaryl groups. Incertain embodiments, the heteroaryl, or substituted heteroaryl groupscomprise nitrogen, oxygen, or sulfur as heteroatoms.

In formula (I), R¹, R², R³, and R⁴ are optional and are independentlyselected from hydrogen, alkylene, substituted alkylene, alkenylene,substituted alkenylene, alkynylene, substituted alkynylene, alkoxy,substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl,aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino,substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, andthioalkoxy.

In certain embodiments, R¹, R², R³, or R⁴ is hydrogen. In certainembodiments, R¹, R², R³, or R⁴ is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹, R², R³, or R⁴ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹, R², R³, or R⁴ is aryl orsubstituted aryl. In certain embodiments, R¹, R², R³, or R⁴ is carboxylester, acyl, acylamino, aminoacyl, aminocarbonylamino, or acyloxy. Incertain embodiments, R¹, R², R³, or R⁴ is aminosulfonyl, sulfonylamino,sulfonyl, sulfonyloxy, or thioalkoxy. In certain embodiments, R¹, R²,R³, or R⁴ is amino or substituted amino.

In formula (I), Y¹, Y², Y³, and Y⁴ are independently selected fromhydrogen; halogen; a moiety that comprises a reactive group thatfacilitates covalent attachment of a molecule of interest; and amolecule of interest. In certain embodiments, Y¹, Y², Y³, or Y⁴ ishydrogen. In certain embodiments, Y¹, Y², Y³, or Y⁴ is halogen. Incertain embodiments, Y¹, Y², Y³, or Y⁴ is a moiety that comprises areactive group that facilitates covalent attachment of a molecule ofinterest. In certain embodiments, Y¹, Y², Y³, or Y⁴ is a molecule ofinterest.

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula or salt thereof:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula or salt thereof:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula or salt thereof:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula:

In certain embodiments, the pi-electrophile reactive partner is a metalbis(dithiolene) compound of the formula:

Azo Compounds

The embodiments provide azo compounds; and compositions comprising thecompounds. A subject azo compound is a compound of the formula:

-   -   wherein    -   Ar¹ is an optional aryl or substituted aryl group;    -   R¹ is optional and is selected from alkylene, substituted        alkylene, alkenylene, substituted alkenylene, alkynylene,        substituted alkynylene, alkoxy, substituted alkoxy, aryl,        substituted aryl, acyl, acylamino, aminoacyl,        aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino,        amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy,        and thioalkoxy; and    -   Y¹ is selected from a moiety that comprises a reactive group        that facilitates covalent attachment of a molecule of interest;        and a molecule of interest.

The embodiments provide azo compounds; and compositions comprising thecompounds. A subject azo compound is a compound of the formula:

-   -   wherein    -   R¹ is optional and is selected from alkylene, substituted        alkylene, alkenylene, substituted alkenylene, alkynylene,        substituted alkynylene, alkoxy, substituted alkoxy, aryl,        substituted aryl, acyl, acylamino, aminoacyl,        aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino,        amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy,        and thioalkoxy;    -   Y¹ is selected from a moiety that comprises a reactive group        that facilitates covalent attachment of a molecule of interest;        and a molecule of interest;    -   R² is selected from alkyl, substituted alkyl, hydroxy, alkoxy,        substituted alkoxy, amino, substituted amino, aminocarbonyl,        carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, and        trihalomethyl; and    -   n is a number from zero to four.

In formula (II), Ar¹ is an optional aryl or substituted aryl group. Incertain embodiments, Ar¹ is an aryl group. In certain embodiments, Ar¹is a substituted aryl group.

In formula (II), R¹ is optional and is selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy.

In certain embodiments, R¹ is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹ is alkoxy or substituted alkoxy.In certain embodiments, R¹ is aryl or substituted aryl. In certainembodiments, R¹ is carboxyl ester, acyl, acylamino, aminoacyl,aminocarbonylamino, or acyloxy. In certain embodiments, R¹ isaminosulfonyl, sulfonylamino, sulfonyl, sulfonyloxy, or thioalkoxy. Incertain embodiments, R¹ is amino or substituted amino.

In formula (II), Y¹ is selected from a moiety that comprises a reactivegroup that facilitates covalent attachment of a molecule of interest;and a molecule of interest. In certain embodiments, Y¹ is a moiety thatcomprises a reactive group that facilitates covalent attachment of amolecule of interest. In certain embodiments, Y¹ is a molecule ofinterest.

In formula (IIb), R² is selected from alkyl, substituted alkyl, hydroxy,alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl,carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, and trihalomethyl.

In certain embodiments, R² is alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, or trihalomethyl. Incertain embodiments, R² is hydroxy, alkoxy, or substituted alkoxy. Incertain embodiments, R² is amino or substituted amino. In certainembodiments, R² is aminocarbonyl, carboxyl, or carboxyl ester. Incertain embodiments, R² is cyano, halogen, or nitro.

In certain embodiments, the pi-electrophile reactive partner is an azocompound of the formula:

In certain embodiments, the pi-electrophile reactive partner is an azocompound of the formula:

The embodiments provide azo compounds; and compositions comprising thecompounds. A subject azo compound is a compound of the formula:

Y¹—R¹—N═N—R²—Y²  (III),

-   -   wherein    -   R¹ and R² are optional and are independently selected from        hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹ and Y² are independently selected from hydrogen; halogen; a        moiety that comprises a reactive group that facilitates covalent        attachment of a molecule of interest; and a molecule of        interest;    -   wherein at least one of Y¹ and Y² is a moiety that comprises a        reactive group that facilitates covalent attachment of a        molecule of interest or a molecule of interest.

In formula (III), R¹ and R² are optional and is selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy.

In certain embodiments, R¹ or R² is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹ or R² is alkoxy or substitutedalkoxy. In certain embodiments, R¹ or R² is aryl or substituted aryl. Incertain embodiments, R¹ or R² is carboxyl ester, acyl, acylamino,aminoacyl, aminocarbonylamino, or acyloxy. In certain embodiments, R¹ orR² is aminosulfonyl, sulfonylamino, sulfonyl, sulfonyloxy, orthioalkoxy. In certain embodiments, R¹ or R² is amino or substitutedamino.

In formula (III), Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest. In certain embodiments, Y¹ or Y² is hydrogen. In certainembodiments, Y¹ or Y² is halogen. In certain embodiments, Y¹ or Y² is amoiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest. In certain embodiments, Y¹ or Y²is a molecule of interest.

In certain embodiments, the pi-electrophile reactive partner is an azocompound of the formula:

Alkynyl Compounds

The embodiments provide alkynyl compounds; and compositions comprisingthe compounds. A subject alkynyl compound is a compound of the formula:

-   -   wherein    -   R¹ and R² are optional and are independently selected from        hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹ and Y² are independently selected from hydrogen; halogen; a        moiety that comprises a reactive group that facilitates covalent        attachment of a molecule of interest; and a molecule of        interest;    -   wherein at least one of Y¹ and Y² is a moiety that comprises a        reactive group that facilitates covalent attachment of a        molecule of interest or a molecule of interest.

In formula (IV), R¹ and R² are optional and is selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy.

In certain embodiments, R¹ or R² is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹ or R² is alkoxy or substitutedalkoxy. In certain embodiments, R¹ or R² is aryl or substituted aryl. Incertain embodiments, R¹ or R² is carboxyl ester, acyl, acylamino,aminoacyl, aminocarbonylamino, or acyloxy. In certain embodiments, R¹ orR² is aminosulfonyl, sulfonylamino, sulfonyl, sulfonyloxy, orthioalkoxy. In certain embodiments, R¹ or R² is amino or substitutedamino.

In formula (IV), Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest. In certain embodiments, Y¹ or Y² is hydrogen. In certainembodiments, Y¹ or Y² is halogen. In certain embodiments, Y¹ or Y² is amoiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest. In certain embodiments, Y¹ or Y²is a molecule of interest.

In certain embodiments, the pi-electrophile reactive partner is analkynyl compound of the formula:

Alkenyl Compounds

The embodiments provide alkenyl compounds; and compositions comprisingthe compounds. A subject alkenyl compound is a compound of the formula:

-   -   wherein    -   R¹ and R² are optional and are independently selected from        hydrogen, alkylene, substituted alkylene, alkenylene,        substituted alkenylene, alkynylene, substituted alkynylene,        alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,        acylamino, aminoacyl, aminocarbonylamino, acyloxy,        aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl        ester, sulfonyl, sulfonyloxy, and thioalkoxy; and    -   Y¹ and Y² are independently selected from hydrogen; halogen; a        moiety that comprises a reactive group that facilitates covalent        attachment of a molecule of interest; and a molecule of        interest;    -   wherein at least one of Y¹ and Y² is a moiety that comprises a        reactive group that facilitates covalent attachment of a        molecule of interest or a molecule of interest.

In formula (V), R¹ and R² are optional and is selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy.

In certain embodiments, R¹ or R² is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹ or R² is alkoxy or substitutedalkoxy. In certain embodiments, R¹ or R² is aryl or substituted aryl. Incertain embodiments, R¹ or R² is carboxyl ester, acyl, acylamino,aminoacyl, aminocarbonylamino, or acyloxy. In certain embodiments, R¹ orR² is aminosulfonyl, sulfonylamino, sulfonyl, sulfonyloxy, orthioalkoxy. In certain embodiments, R¹ or R² is amino or substitutedamino.

In formula (V), Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest. In certain embodiments, Y¹ or Y² is hydrogen. In certainembodiments, Y¹ or Y² is halogen. In certain embodiments, Y¹ or Y² is amoiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest. In certain embodiments, Y¹ or Y²is a molecule of interest.

Ketone Compounds

The embodiments provide ketone compounds; and compositions comprisingthe compounds. A subject ketone compound is a compound of the formula:

-   -   wherein    -   R¹ is optional and is selected from alkylene, substituted        alkylene, alkenylene, substituted alkenylene, alkynylene,        substituted alkynylene, alkoxy, substituted alkoxy, aryl,        substituted aryl, acyl, acylamino, aminoacyl,        aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino,        amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy,        and thioalkoxy; and    -   R², R³, and R⁴ are independently selected from hydrogen, alkyl,        substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino,        substituted amino, aminocarbonyl, carboxyl, carboxyl ester,        cyano, halogen, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, trihalomethyl, aryl, and substituted aryl;        and    -   Y¹ is selected from a moiety that comprises a reactive group        that facilitates covalent attachment of a molecule of interest;        and a molecule of interest.

The embodiments provide ketone compounds; and compositions comprisingthe compounds. A subject ketone compound is a compound of the formula:

-   -   wherein    -   R¹ is optional and is selected from alkylene, substituted        alkylene, alkenylene, substituted alkenylene, alkynylene,        substituted alkynylene, alkoxy, substituted alkoxy, aryl,        substituted aryl, acyl, acylamino, aminoacyl,        aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino,        amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy,        and thioalkoxy; and    -   R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino,        substituted amino, aminocarbonyl, carboxyl, carboxyl ester,        cyano, halogen, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, and trihalomethyl;    -   each R⁵ is independently selected from hydrogen, alkyl,        substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino,        substituted amino, aminocarbonyl, carboxyl, carboxyl ester,        cyano, halogen, nitro, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, and trihalomethyl; and    -   n is number from one to five;    -   Y¹ is selected from a moiety that comprises a reactive group        that facilitates covalent attachment of a molecule of interest;        and a molecule of interest.

In formula (VI), R¹ is optional and is selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy.

In certain embodiments, R¹ is alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, or substitutedalkynylene. In certain embodiments, R¹ is alkoxy or substituted alkoxy.In certain embodiments, R¹ is aryl or substituted aryl. In certainembodiments, R¹ is carboxyl ester, acyl, acylamino, aminoacyl,aminocarbonylamino, or acyloxy. In certain embodiments, R¹ isaminosulfonyl, sulfonylamino, sulfonyl, sulfonyloxy, or thioalkoxy. Incertain embodiments, R¹ is amino or substituted amino.

In formula (VI), Y¹ is selected from a moiety that comprises a reactivegroup that facilitates covalent attachment of a molecule of interest;and a molecule of interest. In certain embodiments, Y¹ is a moiety thatcomprises a reactive group that facilitates covalent attachment of amolecule of interest. In certain embodiments, Y¹ is a molecule ofinterest.

In formula (VIa), R², R³, and R⁴ are independently selected fromhydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy,amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester,cyano, halogen, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, trihalomethyl, aryl, and substituted aryl.

In certain embodiments, R², R³, or R⁴ is hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, ortrihalomethyl. In certain embodiments, R², R³, or R⁴ is hydroxy, alkoxy,or substituted alkoxy. In certain embodiments, R², R³, or R⁴ is amino orsubstituted amino. In certain embodiments, R², R³, or R⁴ isaminocarbonyl, carboxyl, carboxyl ester, cyano, or halogen. In certainembodiments, R², R³, or R⁴ is aryl or substituted aryl.

In formula (VIb), each R⁵ is independently selected from hydrogen,alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino,substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano,halogen, nitro, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, and trihalomethyl; and n is number from one to five.

In certain embodiments, R⁵ is hydrogen, alkyl, or substituted alkyl. Incertain embodiments, R⁵ is hydroxy, alkoxy, or substituted alkoxy. Incertain embodiments, R⁵ is amino or substituted amino. In certainembodiments, R⁵ is aminocarbonyl, carboxyl, or carboxyl ester. Incertain embodiments, R⁵ is cyano, halogen, or nitro. In certainembodiments, R⁵ is alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, or trihalomethyl.

In formula (VIb), n is number from one to five. In certain embodiments,n is one. In certain embodiments, n is two. In certain embodiments, n isthree. In certain embodiments, n is four. In certain embodiments, n isfive.

In certain embodiments, the pi-electrophile reactive partner is analkynyl compound of the formula:

In certain embodiments, the pi-electrophile reactive partner is analkynyl compound of the formula:

wherein n is a number form 1 to 4.Reactive Group that Facilitates Covalent Attachment of a Molecule ofInterest

In some embodiments, Y is a reactive group. Suitable reactive groupsinclude, but are not necessarily limited to, carboxyl, amine, (e.g.,alkyl amine (e.g., lower alkyl amine), aryl amine), ester (e.g., alkylester (e.g., lower alkyl ester, benzyl ester), aryl ester, substitutedaryl ester), thioester, sulfonyl halide, alcohol, thiol, succinimidylester, isothiocyanate, iodoacetamide, maleimide, hydrazine, and thelike. In some embodiments, Y is a reactive group selected from acarboxyl, an amine, an ester, a thioester, a sulfonyl halide, analcohol, a thiol, a succinimidyl ester, an isothiocyanate, aniodoacetamide, a maleimide, and a hydrazine.

Other suitable reactive groups include, but are not necessarily limitedto, aminooxy, aldehyde, ketone, nitrile oxide, nitrone, tetrazine,azirine, tetrazole, alkene, alkyne, cyclooctyne, trans-cyclooctene,norbornene, and azide.

Molecules of Interest

In some embodiments, Y is a molecule of interest. Suitable molecules ofinterest include, but are not limited to, a detectable label; a toxin(including cytotoxins); a peptide; a drug; a member of a specificbinding pair; an epitope tag; and the like.

Where Y is a molecule of interest, the pi-electrophile comprises amolecule desired for delivery to a quadricyclane-containing biomoleculevia [2+2+2] reaction. Molecules of interest that may be desirable fordelivery include, but are not necessarily limited to, detectable labels(e.g., spin labels, fluorescence resonance energy transfer (FRET)-typedyes, e.g., for studying structure of biomolecules in vivo); smallmolecule drugs; cytotoxic molecules (e.g., drugs); ligands for bindingby a target receptor (e.g., to facilitate viral attachment; attachmentof a targeting protein present on a liposome, etc.); tags to aid inpurification by, for example, affinity chromatography (e.g., attachmentof a FLAG epitope); members of specific binding pairs (e.g., biotin,where the specific binding pair is biotin and avidin); molecules tofacilitate selective attachment of the polypeptide to a surface; and thelike. Specific, non-limiting examples are provided below.

Detectable Labels

The compositions and methods of the present disclosure can be used todeliver a detectable label to a quadricyclane-containing biomolecule.Thus, in some embodiments, a pi-electrophile comprises a detectablelabel, covalently bound to the pi-electrophile either directly orthrough a linker.

Exemplary detectable labels include, but are not necessarily limited to,fluorescent molecules (e.g., autofluorescent molecules, molecules thatfluoresce upon contact with a reagent, etc.), radioactive labels (e.g.,¹¹¹In, ¹²⁵I, ¹³¹I, ²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like); a positron emissiontomography (PET) imaging label (e.g. ¹⁸F); fluorescent tags; imagingreagents (e.g., those described in U.S. Pat. No. 4,741,900 and U.S. Pat.No. 5,326,856), and the like. Detectable labels also include peptides orpolypeptides that can be detected by antibody binding, e.g., by bindingof a detectably labeled antibody or by detection of bound antibodythrough a sandwich-type assay. Also suitable for use are quantum dots(e.g., detectably labeled semiconductor nanocrystals, such asfluorescently labeled quantum dots, antibody-conjugated quantum dots,and the like). See, e.g., Dubertret et al. 2002 Science 298:759-1762;Chan et al. (1998) Science 281:2016-2018; U.S. Pat. No. 6,855,551;Bruchez et al. (1998) Science 281:2013-2016.

Suitable fluorescent molecules (fluorophores) include, but are notlimited to, fluorescein, fluorescein isothiocyanate, succinimidyl estersof carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM(tetramethylrhodamine-, methyl ester), TMRE (tetramethylrhodamine, ethylester), tetramethylrosamine, rhodamine B and4-dimethylaminotetramethylrosamine, green fluorescent protein,blue-shifted green fluorescent protein, cyan-shifted green fluorescentprotein, red-shifted green fluorescent protein, yellow-shifted greenfluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes, a DDAOcompound (e.g., 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one), cascade blue, and thelike. Fluorophores of interest are further described in WO 01/42505 andWO 01/86001.

Suitable fluorescent proteins and chromogenic proteins include, but arenot limited to, a green fluorescent protein (GFP), including, but notlimited to, a GFP derived from Aequoria victoria or a derivativethereof, e.g., a “humanized” derivative such as Enhanced GFP, which isavailable commercially, e.g., from Clontech, Inc.; a GFP from anotherspecies such as Renilla reniformis, Renilla mulleri, or Ptilosarcusguernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J.Protein Chem. 20:507-519; “humanized” recombinant GFP (hrGFP)(Stratagene); any of a variety of fluorescent and colored proteins fromAnthozoan species, as described in, e.g., Matz et al. (1999) NatureBiotechnol. 17:969-973; and the like.

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., CYPYDVPDYA; SEQ ID NO:1), FLAG (e.g., DYKDDDDK; SEQ ID NO:2),FLAG-C (e.g., DYKDDDDKC; SEQ ID NO:3, c-myc (e.g., CEQKLISEEDL; SEQ IDNO:4), a metal ion affinity tag such as a polyhistidine tag (e.g.,His₆), and the like.

Suitable imaging agents include positive contrast agents and negativecontrast agents. Suitable positive contrast agents include, but are notlimited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA);Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA);Gadolinium-1,4,7-tris(carbonylmethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane(Gd-HP-DO3A); Manganese(II)-dipyridoxal diphosphate (Mn-DPDP);Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); andthe like. Suitable negative contrast agents include, but are not limitedto, a superparamagnetic iron oxide (SPIO) imaging agent; and aperfluorocarbon, where suitable perfluorocarbons include, but are notlimited to, fluoroheptanes, fluorocycloheptanes,fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes,fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes,fluorodimethylcyclopentanes, fluoromethylcyclobutanes,fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes,fluorocyclobutanse, fluoropropanes, fluoroethers, fluoropolyethers,fluorotriethylamines, perfluorohexanes, perfluoropentanes,perfluorobutanes, perfluoropropanes, sulfur hexafluoride, and the like.

Suitable imaging agents also include spin labels suitable for use inelectron paramagnetic resonance (EPR) and dynamic nuclear polarization.

Specific Binding Partners

In another embodiment, a pi-electrophile comprises a member of a pair ofbinding partners. A member of a pair of binding partners is referred toherein as a “specific binding partner.”

Suitable specific binding partners include, but are not limited to, amember of a receptor/ligand pair; a member of an antibody/antigen pair;a member of a lectin/carbohydrate pair; a member of an enzyme/substratepair; biotin/avidin; biotin/streptavidin; digoxin/antidigoxin; and thelike. Suitable specific binding partners include, but are not limited toa receptor ligand; a receptor for a ligand; a ligand-binding portion ofa receptor; an antibody; an antigen-binding fragment of an antibody; anantigen; a hapten; a lectin; a lectin-binding carbohydrate; an enzymesubstrate; an irreversible inhibitor of an enzyme (e.g., an irreversibleinhibitor that binds a substrate binding site of an enzyme, e.g., a“suicide” substrate); and the like.

Suitable ligand members of receptor/ligand pairs include, but are notlimited to, neurotransmitters such as opioid compounds, acetylcholine,and the like; viral proteins that bind to a cell surface receptor, e.g.,human immunodeficiency virus gp120, and the like; hormones; and thelike.

Suitable antigen-binding antibody fragments include F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv, and Fd fragments, single-chain antibodies, andfusion proteins comprising an antigen-binding portion of an antibody anda non-antibody protein (e.g., an antigen-binding fragment of an antibodyfused to an immunoglobulin constant region).

Suitable haptens include, but are not limited to,(4-hydroxy-3-nitrophenyl) acetyl; diethylenetriaminepentaacetic acid(DTPA) or one of its metal complexes; paranitrophenyl; biotin;fluorescein isothiocyanate; and the like.

Drugs

Suitable drugs that can be attached to a pi-electrophile include, butare not limited to, cytotoxic compounds (e.g., cancer chemotherapeuticcompounds); antiviral compounds; biological response modifiers (e.g.,hormones, chemokines, cytokines, interleukins, etc.); microtubuleaffecting agents; hormone modulators; steroidal compounds; and the like.

Suitable cancer chemotherapeutic compounds include, but are not limitedto, non-peptidic (i.e., non-proteinaceous) compounds that reduceproliferation of cancer cells; peptidic compounds that reduceproliferation of cancer cells; anti-metabolite agents; cytotoxic agents;and cytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (vinca) alkaloids, and steroid hormones.

Suitable agents that act to reduce cellular proliferation include, butare not limited to, alkylating agents, such as nitrogen mustards,nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes,including, but not limited to, mechlorethamine, cyclophosphamide(Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine(CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracilmustard, chlormethine, ifosfamide, chlorambucil, pipobroman,triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Suitable antimetabolite agents include folic acid analogs, pyrimidineanalogs, purine analogs, and adenosine deaminase inhibitors, including,but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside,fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine(6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable anti-proliferative natural products and their derivatives,(e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, andepipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel(Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C,L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine,vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g.etoposide, teniposide, etc.; antibiotics, e.g. anthracycline,daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine),idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.;phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides,e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506(tacrolimus, prograf), rapamycin, etc.; and the like.

Other suitable anti-proliferative cytotoxic agents are navelbene,CPT-11, anastrazole, letrazole, capecitabine, reloxafine,cyclophosphamide, ifosamide, and droloxafine.

Suitable microtubule affecting agents that have antiproliferativeactivity include, but are not limited to, allocolchicine (NSC 406042),Halichondrin B (NSC 609395), colchicine (NSC 757), colchicinederivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine(NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol®derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysterin, vinblastine sulfate, vincristine sulfate, natural andsynthetic epothilones including but not limited to, eopthilone A,epothilone B, discodermolide; estramustine, nocodazole, and the like.

Suitable hormone modulators and steroids (including synthetic analogs)include, but are not limited to, adrenocorticosteroids, e.g. prednisone,dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesteronecaproate, medroxyprogesterone acetate, megestrol acetate, estradiol,clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol,testosterone, fluoxymesterone, dromostanolone propionate, testolactone,methylprednisolone, methyl-testosterone, prednisolone, triamcinolone,chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil),Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferationand differentiation, therefore compounds that bind to the estrogenreceptor are used to block this activity. Corticosteroids may inhibit Tcell proliferation.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are also suitable for attachment to a pi-electrophile. “Taxanes”include paclitaxel, as well as any active taxane derivative or pro-drug.“Paclitaxel” (which should be understood herein to include analogues,formulations, and derivatives such as, for example, docetaxel, TAXOL™,TAXOTERE™ (a formulation of docetaxel), 10-desacetyl analogs ofpaclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers that are suitable for attachment to a pielectrophile moiety include, but are not limited to, (1) inhibitors oftyrosine kinase (RTK) activity; (2) inhibitors of serine/threoninekinase activity; (3) tumor-associated antigen antagonists, such asantibodies that bind specifically to a tumor antigen; (4) apoptosisreceptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ (8)colony-stimulating factors; and (9) inhibitors of angiogenesis.

Compositions

The present disclosure further provides compositions, includingpharmaceutical compositions, comprising a pi-electrophile comprising amolecule of interest. A subject composition generally comprises api-electrophile comprising a molecule of interest; and at least oneadditional compound. Suitable additional compounds include, but are notlimited to: a salt, such as a magnesium salt, a sodium salt, etc., e.g.,NaCl, MgCl₂, KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

In some embodiments, a subject composition comprises a pi-electrophilecomprising a molecule of interest; and a pharmaceutically acceptableexcipient. A wide variety of pharmaceutically acceptable excipients isknown in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy,” 20^(th) edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Quadricyclane Reaction Partner

The present disclosure provides quadricyclane-modified biomolecules; andcompositions comprising the quadricyclane-modified biomolecules. Asubject quadricyclane-modified biomolecule is a compound of the formula:

-   -   wherein    -   R¹, R², R³, R⁴, and R⁵ are optional linkers;    -   Z¹, Z², Z³, Z⁴, and Z⁵ are independently selected from hydrogen        and a biomolecule;    -   wherein at least one of Z¹, Z², Z³, Z⁴, and Z⁵ is a biomolecule.

Exemplary biomolecules include, e.g., amino acids; peptides (e.g.,having from about 2 to about 10 amino acids); monosaccharides;oligosaccharides; fatty acids; lipids; nucleotides; oligonucleotides;enzyme substrates; enzyme inhibitors; sterols; co-factors; Co-Aderivatives; and the like.

Suitable linkers for R¹, R², and R³ include, but are not limited to,alkylene, substituted alkylene, alkenylene, substituted alkenylene,alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl,substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino,acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino,carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy.

Biomolecules Attached to Quadricyclane

Exemplary biomolecules include, e.g., amino acids; peptides (e.g.,having from about 2 to about 10 amino acids); monosaccharides;oligosaccharides; fatty acids; lipids; nucleotides; oligonucleotides;enzyme substrates; enzyme inhibitors; sterols; co-factors; Co-Aderivatives; and the like. Thus, the present disclosure providescompounds comprising a biomolecule linked, directly or via a linker, toa quadricyclane moiety. Thus, e.g., the present disclosure providespeptide-quadricyclane conjugates, lipid-quadricyclane conjugates,sugar-quadricyclane conjugates, nucleotide-quadricyclane conjugates,etc.

The biomolecules can be naturally occurring, or may be synthetically orrecombinantly produced, and may be isolated, substantially purified, orpresent within the native milieu of the unmodified molecule upon whichthe quadricyclane-containing biomolecule is based (e.g., on a cellsurface or within a cell, including within a host animal, e.g., amammalian animal, such as a murine host (e.g., rat, mouse), hamster,canine, feline, bovine, swine, and the like). In some embodiments, thebiomolecule is present in vitro in a cell-free reaction. In otherembodiments, the biomolecule is present in a cell and/or displayed onthe surface of a cell. In many embodiments of interest, the biomoleculeis in a living cell; on the surface of a living cell; in a livingorganism, e.g., in a living multicellular organism. Suitable livingcells include cells that are part of a living multicellular organism;cells isolated from a multicellular organism; immortalized cell lines;and the like.

Where the biomolecule is a peptide or a polypeptide, the peptide orpolypeptide may be composed of D-amino acids, L-amino acids,non-naturally-occurring amino acids (e.g., non-coded amino acids), ortwo or more of the foregoing, and may be further modified, eithernaturally, synthetically, or recombinantly, to include other moieties.For example, a polypeptide may be a lipoprotein, a glycoprotein, orother such modified protein.

In general, the biomolecule comprises at least one quadricyclane forreaction with pi-electrophile, but may comprise 2 or more, 3 or more, 5or more, 10 or more quadricyclanes. The number of quadricyclanes thatmay be present in a biomolecule will vary according to the intendedapplication of the final product of the reaction, the nature of thebiomolecule itself, and other considerations which will be readilyapparent to the ordinarily skilled artisan.

This embodiment is particularly useful in modification of a biomoleculein vivo. In this embodiment, the biomolecule is modified to comprise aquadricyclane at the point at which linkage to the pi-electrophile isdesired. For example, where the biomolecule is a polypeptide, thepolypeptide is modified to contain a quadricyclane at an N-terminus, atthe C-terminus, or at an internal amino acid within the polypeptide.Where the biomolecule is a glycoprotein, a sugar residue of theglycoprotein can be modified to contain a quadricyclane.

Quadricyclane-Pi Electrophile Ligation Reaction

The present disclosure provides methods for chemoselective modificationof a biomolecule comprising a quadricyclane. The methods generallyinvolve reacting a quadracyclane in a quadricyclane-containingbiomolecule with a pi-electrophile comprising a molecule of interest.Thus, after a [2+2+2] cycloaddition reaction of the quadricyclane andpi-electrophile, the molecule of interest is covalently bound to thebiomolecule.

In many embodiments, a subject method for synthetically modifying acellular component generally involves: introducing a quadricyclanemoiety into a cellular component, thereby generating aquadricyclane-modified cellular component; and contacting the cellcomprising the quadricyclane-modified cellular component with a reactionpartner comprising a pi-electrophile comprising a molecule of interest,the contacting being under physiological conditions. The contacting stepresults in reaction between the quadricyclane group ofquadricyclane-modified cellular component and the pi-electrophile of thereaction partner, thereby synthetically and covalently modifying thecellular component. In some embodiments, the method is carried out in acell-free system in vitro. In some embodiments, the method is carriedout on living cells in vitro. In other embodiments, the method iscarried out on living cells ex vivo. In still other embodiments, themethod is carried out on living cells in vivo.

In one embodiment, the chemoselective ligation is designed for use infully aqueous, physiological conditions and involves production of astable, final product comprising a fused quadracyclane/pi-electrophile.In general, this embodiment involves reacting a first reactantcomprising a pi-electrophile comprising a molecule of interest with asecond reactant comprising a quadricyclane-modified biomolecule, suchthat a covalent bond is formed between the first and second reactants byreaction of the pi-electrophile with the quadricyclane.

A subject ligation reaction can be carried out sequentially, orsimultaneously, with other ligation reactions. For example, a subjectquadricyclane-pi electrophile based ligation reaction can be carried outsequentially or simultaneously with a reaction based on a modifiedphosphine-azide ligation reaction. See, e.g., U.S. Pat. No. 6,570,040.As another example, a subject quadricyclane-pi electrophile basedligation reaction can be carried out sequentially or simultaneously witha reaction based on a modified cycloalkyne-azide ligation reaction. See,e.g., U.S. Pat. No. 7,808,619; and U.S. Patent Publication No.2009/0068738. In some cases, a subject ligation reaction can be carriedout substantially simultaneously with both a modified phosphine-azideligation reaction and/or a modified cycloalkyne-azide ligation reaction.As an example, the pi-electrophile can comprise a first molecule ofinterest (e.g., a first Y group); the modified phosphine can comprise asecond molecule of interest that is different from the first molecule ofinterest; and the modified cycloalkyne can comprise a third molecule ofinterest that is different from the first and the second molecule ofinterest. For example, the first, second, and third molecules ofinterest can be different detectable labels (e.g., different dyes;etc.).

In certain embodiments, a subject quadricyclane-pi electrophile basedligation reaction can be carried out sequentially or simultaneously withother reactions such as use of carbonyl chemistry (such as, but notlimited to, condensation reaction between aldehydes and aminooxycompounds), Staudinger ligation (reaction of an azide with a phosphineor phosphite to produce an iminophosphorane), and cyclooctyne chemistry(such as cycloaddition of cyclooctyne with an azide; see, e.g., U.S.Patent Publication No. 2009-0068738-A1).

Utility

Subject pi-electrophiles comprising molecules of interest, and subjectmodification methods, are useful in a variety of applications, includingresearch applications and diagnostic applications.

Bioorthogonal Chemistry

New additions to the bioorthogonal chemistry compendium can advancebiological research by enabling multiplexed analysis of biomolecules incomplex systems. The quadricyclane ligation can be used as a newbioorthogonal reaction. This reaction has a second-order rate constantof 0.25 M⁻¹ s⁻¹, on par with fast bioorthogonal reactions of azides, andproceeds readily in aqueous environments. The quadricyclane ligation iscompatible with, and orthogonal to, strain-promoted azide-alkynecycloaddition and oxime ligation chemistries.

This quadricyclane-pi-electrophile is orthogonal to a current cohort.The published bioorthogonal transformations represent four broadreaction types: 1,3-dipolar cycloadditions, Diels-Alder reactions,metal-catalyzed couplings, and nucleophilic additions. Outside of thisspace lies the [2+2+2] cycloaddition reaction, a popular choice for therapid assembly of functionalized ring systems. In practice, suchreactions are typically metal catalyzed as a means to overcome anotherwise significant entropic barrier. However, the highly strainedhydrocarbon quadricyclane directly undergoes [2+2+2] cycloaddition withspecific types of π systems.

Research Applications

In some embodiments, the pi-electrophile compounds, and subjectmodification methods, are useful in research applications. Applicationsof interest include research applications, e.g., exploring functionaland physical characteristics of a receptor; proteomics; metabolomics;and the like. Research applications also include drug discovery or otherscreening applications.

Proteomics aims to detect, identify, and quantify proteins to obtainbiologically relevant information. Metabolomics is the detection,identification, and quantification of metabolites and other smallmolecules such as lipids and carbohydrates. Fiehn (2001) Comparative andFunctional Genomics 2:155-168; and U.S. Pat. No. 6,873,914.

Drug discovery applications include, but are not limited to, identifyingagents that inhibit cancer cell viability and/or growth. Thus, in someembodiments, the instant disclosure provides methods of identifying anagent that inhibits cancer cell viability and/or growth. The methodsgenerally involve modifying a component of the cell to comprise aquadricyclane-modified biomolecule; contacting the cell, in the presenceof a test agent, with a reaction partner comprising a pi-electrophilecomprising a molecule of interest, the contacting being underphysiological conditions; where the contacting results in reactionbetween the quadricyclane group and the pi-electrophile, therebysynthetically and covalently modifying the cellular component; anddetermining the effect, if any, of the test agent on the level ofmodification of the cell with the pi-electrophile comprising a moleculeof interest.

Where the cancer cell is one that produces a higher amount of acarbohydrate than a normal (non-cancerous) cell of the same cell type,the method provides for identifying an agent that reduces growth and/orviability of the cancerous cell.

Diagnostic and Therapeutic Applications

Applications of interest also include diagnostic applications, e.g., fordetection of cancer; and the like, where a pi-electrophile compoundcomprising a detectable label is used to label a quadricyclane-modifiedbiomolecule, e.g., a quadricyclane-labeled biomolecule present on acancer cell. Applications of interest also include therapeuticapplications, where a drug or other therapeutic agent is delivered to aquadricyclane-modified biomolecule, using a pi electrophile compoundthat comprises a covalently linked drug or other therapeutic agent.

In some embodiments, a subject method is used for in vivo imaging, e.g.,to determine the metabolic or other state of a cell in an organism,e.g., an individual. As one non-limiting example, a subject method canbe applied to in vivo imaging of cancer cells in an individual (e.g., amammal, including rodents, lagomorphs, felines, canines, equines,bovines, ovines, caprines, non-human primates, and humans).

One exemplary, non-limiting application of a subject quadracyclane-pielectophile cycloaddition is in the detection of metabolic change incells that occur as they alter their phenotype. As one example, alteredglycosylation patterns are a hallmark of the tumor phenotype, comprisingunder- and over-expression of naturally-occurring glycans as well as thepresentation of glycans normally restricted to expression duringembryonic development. Examples of common antigens associated withtransformed cells are sialyl Lewis a, sialyl Lewis x, sialyl T, sialylTn, and polysialic acid (PSA). Jørgensen et al. (1995) Cancer Res. 55,1817-1819; Sell (1990) Hum. Pathology 21, 1003-1019; Taki et al. (1988)J. Biochem. 103, 998-1003; Gabius (1988) Angew. Chem. Int. Ed. Engl. 27,1267-1276; Feizi (1991) Trends Biochem. Sci. 16, 84-86;Taylor-Papadimitriou and Epenetos (1994) Trends Biotech. 12, 227-233;Hakomori and Zhang (1997) Chem. Biol. 4, 97-104; Dohi et al. (1994)Cancer 73, 1552. These antigens share an important feature—they eachcontain terminal sialic acid. PSA is a homopolymer of sialic acidresidues up to 50 units in length. Elevated levels of sialic acid arehighly correlated with the transformed phenotype in many cancers,including gastric (Dohi et al. (1994) Cancer 73, 1552; and Yamashita etal. (1995) J. Natl. Cancer Inst. 87, 441-446), colon (Yamashita et al.(1995) J. Natl. Cancer Inst. 87, 441-446; Hanski et al. (1995) CancerRes. 55, 928-933; Hanski et al. (1993) Cancer Res. 53, 4082-4088; Yanget al. (1994) Glycobiology 4, 873-884; Saitoh et al. (1992) J. Biol.Chem. 267, 5700-5711), pancreatic (Sawada et al. (1994) Int. J. Cancer57, 901-907), liver (Sawada et al. (1994) J. Biol. Chem. 269,1425-1431), lung (Weibel et al. (1988) Cancer Res. 48, 4318-4323),prostate (Jørgensen et al. (1995) Cancer Res. 55, 1817-1819), kidney(Roth et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2999-3000), andbreast cancers (Cho et al. (1994) Cancer Res. 54, 6302-6305), as well asseveral types of leukemia (Joshi et al. (1987) Cancer Res. 47,3551-3557; Altevogt et al. (1983) Cancer Res. 43, 5138-5144; Okada etal. (1994) Cancer 73, 1811-1816). A strong correlation between the levelof cell surface sialic acid and metastatic potential has also beenobserved in several different tumor types (Kakeji et al. (1995) Brit. J.Cancer 71, 191-195; Takano et al. (1994) Glycobiology 4, 665-674). Thecollective display of multiple sialylated antigens on a single cancercell can account for the fact that so many different tumor types sharethe high sialic acid phenotype without necessarily expressing anidentical complement of antigens (Roth et al. (1988) supra).Consequently, diagnostic or therapeutic strategies that target cells onthe basis of sialic acid levels have broad applicability to manycancers.

Introduction and incorporation of unnatural quadricyclane sugars(ManNQu, GalNQu) into living animals provides for detection of changesin metabolic state. Via the attachment of the appropriate epitope tag,the pi-electrophile compound labels these cells in a living organism,and consequently detects changes in metabolic state. Early detection oftumorigenic cells and subsequent intervention reduces the severity andincreases survival rates for cancer patients.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Syntheses of Compounds General Experimental Procedure

All chemical reagents were purchased from Sigma-Aldrich, Acros or TCIand used without purification unless noted otherwise. Anhydrousdimethylformamide (DMF) and methanol (MeOH) were purchased from Aldrichor Acros in sealed bottles; all other solvents were purified asdescribed by Pangborn et al. (Pangborn, A. B.; Giardello, M. A.; Grubbs,R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518.)Solvent was removed by reduced pressure with a Buchi Rotovapor R-114equipped with a Welch self-cleaning dry vacuum. Products were furtherdried by reduced pressure with an Edwards RV5 high vacuum.Lyophilization was performed on a LABCONCO FreeZone® instrument equippedwith an Edwards RV2 pump. Thin layer chromatography was performed withEMD 60 Å silica gel plates. Flash chromatography was performed usingSilicycle® 60 Å 230-400 mesh silica. All ¹H and ¹³C spectra are reportedin ppm and referenced to solvent peaks. Spectra were obtained on BrukerAVQ-400, AVB-400, DRX-500, AV-500, or AV-600 instruments. UV/Vis/NIRspectra were acquired on a CARY 100 Bio UV-Visible Spectrophotometerwith a range of 200-900 nm. Electron impact (EI) and electrosprayionization (ESI) mass spectra were obtained from the UC Berkeley MassSpectrometry Facility. High performance liquid chromatography (HPLC) wasperformed on a Varian Pro Star or Varian Prep Star instrument with a C18column.

Experimental Procedures

Complex 3.

Bis(dithiobenzil)nickel(II) (2, 50 mg, 0.092 mmol, 1.0 equiv.) wascombined with 7-acetoxy quadricyclane 1 (30 mg, 0.20 mmol, 2.2 equiv.)in dichloromethane (1 mL) for 5 days in the dark. The reaction wasevaporated to ˜200 mL and methanol (1 mL) was added. This mixture wasplaced in the fridge until brown precipitate formed. The precipitate wascollected and washed with minimal amounts of methanol to yield 40 mg of3 (0.058 mmol, 63%) Some product was left in the mother liquor. Thecrude ¹H nuclear magnetic resonance (NMR) showed ˜90% conversion of 2 to3. R_(f)=0.1 in 1:3 hexanes/dichloromethane. ¹H NMR (600 MHz, CDCl₃): δ7.28-7.26 (m, 4H), 7.19-7.10 (m, 16H), 5.63 (s, 2H), 4.97 (s, 1H), 4.04(s, 2H), 2.43 (apparent q, J=1.7 Hz, 2H), 1.97 (s, 3H). ¹³C NMR (125MHz, CDCl₃): δ 170.8, 162.2, 140.1, 137.4, 133.0, 129.9, 129.1, 128.7,128.4, 128.1, 127.5, 118.0, 85.4, 62.0, 53.6, 49.5, 21.1. HRMS (EI):calcd. for C₃₇H₂₀O₂NiS₄ ⁺ [M]⁺, 692.0482; found, 692.0485.

Methyl 4-(phenylethynyl)benzoate (6)

Phenyl acetylene (1.5 mL, 14 mmol, 1.3 equiv.) and methyl-4-iodobenzoate(3.0 g, 11 mmol, 1.0 equiv.) were dissolved in tetrahydrofuran (THF) (90mL, anhydrous). To this solution, CuI (300 mg, 1.5 mmol, 0.14 equiv.),PdCl₂(PPh₃)₂ (450 mg, 0.64 mmol, 0.06 equiv), and NEt₃ (6.0 mL, 43 mmol,3.9 equiv.) were added. Upon NEt₃ addition, the reaction mixture turneddark black. The mixture was stirred at room temperature until thesolution was no longer dark (˜2 h), at which point the reaction wasquenched with methanol (25 mL), evaporated to dryness, and purified bysilica gel chromatography with hexanes/ethyl acetate (200:1, 100:1,50:1, 25:1, 10:1, 8:1, 6:1). This procedure yielded pure 6 in 99% yield(2.7 g, 11 mmol). R_(f)=0.5 in 8:1 hexanes/ethyl acetate. ¹H NMR (400MHz, CDCl₃): δ 8.02 (d, J=8.1 Hz, 2H), 7.59 (d, J=8.4 Hz, 2H), 7.56-7.54(m, 2H), 7.38-7.36 (m, 3H), 3.93 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ166.8, 131.9, 131.7, 129.7, 129.7, 129.0, 128.6, 128.2, 122.9, 92.6,88.8, 52.4. HRMS (EI): calcd. for C₁₆H₁₂O₂ ⁺ [M]⁺, 236.0837; found,236.0841.

Methyl 4-(2-oxo-5-phenyl-1,3-dithiol-4-yl)benzoate (8)

Methyl 4-(phenylethynyl)benzoate 6 (250 mg, 1.1 mmol, 1.0 equiv.) wascombined with diisopropyl xanthogen disulfide 7 (290 mg, 1.1 mmol, 1.0equiv.) and 1,1′-azobis(cyclohexanecarbonitrile) (110 mg, 0.48 mmol,0.45 equiv.) in m-xylene (2.2 mL, anhydrous). The mixture was heated toreflux for 20 h, at which point the reaction mixture was evaporated todryness and 8 was purified by silica gel chromatography eluting withhexanes/ethyl acetate (40:1). This procedure resulted in 130 mg of 8(0.40 mmol, 37%). R_(f)=0.5 in 6:1 hexanes/ethyl acetate. ¹H NMR (600MHz, CDCl₃): δ 7.91 (d, J=7.1 Hz, 2H), 7.31-7.30 (m, 1H), 7.29-7.26 (m,4H), 7.20-7.19 (m, 2H), 3.90 (s, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 190.2,166.5, 136.5, 131.6, 131.0, 130.4, 130.2, 129.8, 129.7, 129.4, 129.2,127.6, 52.5. HRMS (EI): calcd. for C₁₇H₁₂O₃S₂ ⁺ [M]⁺, 328.0228; found,328.0222.

4-(2-oxo-5-(4-sulfophenyl)-1,3-dithiol-4-yl)benzoic acid (9)

Compound 8 (700 mg, 2.1 mmol, 1.0 equiv.) was dissolved in sulfuric acid(8 mL, 18 M) and fuming sulfuric acid (80 μL, 20% in H₂SO₄) was added.This mixture was heated to 90° C. overnight. The following day it wascooled to room temperature, neutralized with NaOH and NaHCO₃ andevaporated. The resulting solid was subjected to methanol (˜100 mL) andsonicated. This solution was then filtered and the filtrate wasevaporated to dryness. The remaining solid was purified by silica gelchromatography with an acetonitrile/methanol solvent system (15:1, 10:1)to result in 620 mg of pure 9 (1.6 mmol, 74%). R_(f)=0.5 in 9:1acetonitrile/water. ¹H NMR (600 MHz, MeOD): δ 7.93 (d, J=8.4 Hz, 2H),7.76 (d, J=8.4 Hz, 2H), 7.35 (dd, J=13.9, 8.4 Hz, 4H). ¹³C NMR (150 MHz,MeOD): δ 190.9, 169.2, 147.1, 137.1, 134.6, 132.9, 131.3, 130.9, 130.85,130.4, 130.0, 127.8. HRMS (ESI): calcd. for C₁₆H₉O₆S₃ ⁻ [M−H]⁻ 392.9567;found 392.9565.

4-(5-(4-(isopropylcarbamoyl)phenyl)-2-oxo-1,3-dithiol-4-yl)benzenesulfonicacid (10)

Compound 9 (130 mg, 0.33 mmol, 1.0 equiv.) was dissolved indimethylformamide (5 mL, anhydrous). To this solution, isopropyl amine(30 μL, 0.037 mol, 1.1 equiv.),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl, 96mg, 0.50 mmol, 1.5 equiv.), hydroxybenzotriazole hydrate (HOBt, 65 mg,0.42 mmol, 1.3 equiv.), and NEt₃ (90 μL, 0.65 mmol, 1.9 equiv.) wereadded. The mixture was stirred at room temperature for 5 h, at whichpoint it was evaporated to dryness. Compound 10 was purified by HPLCusing a water/methanol solvent system with a gradient of 30-95% methanolover 25 min. The desired product elutes at 14 min. This procedureresulted in pure 10 (56 mg, 0.13 mmol, 39%). ¹H NMR (600 MHz, MeOD): δ7.75-7.72 (m, 4H), 7.34-7.31 (m, 4H), 4.17 (sep, J=6.6 Hz, 1H), 1.22 (d,J=6.6 Hz, 6H). ¹³C NMR (150 MHz, MeOD): δ 191.0, 168.5, 147.3, 136.8,135.8, 134.7, 131.0, 130.9, 130.3, 130.1, 129.1, 127.8, 43.4, 22.6. HRMS(ESI): calcd. for C₁₉H₁₆O₅N₁S₃ ⁻ [M−H]⁻ 434.0196; found, 434.0193.

Ni bis(dithiolene) 14

4-(5-(4-(Isopropylcarbamoyl)phenyl)-2-oxo-1,3-dithiol-4-yl)benzenesulfonicacid 10 (45 mg, 0.10 mmol, 1.0 equiv.) was dissolved in a mixture ofTHF/MeOH (1 mL/0.7 mL). Tetramethyl ammonium hydroxide pentahydrate (40mg, 0.22 mmol, 2.2 equiv.) was dissolved in MeOH (0.2 mL) and added tothe solution with compound 10. The mixture turned a light orange color.After 30 min, NiCl₂.6H₂O (12 mg, 0.050 mmol, 0.50 equiv.) was added andthe mixture turned dark red. This mixture was stirred at roomtemperature overnight. The following morning, iodine (12 mg, 0.047 mmol,0.051 equiv.) was added and the mixture became dark blue. After 2 hstirring at room temperature, the mixture was evaporated to dryness andpurified by silica gel chromatography eluting with acetonitrile/water(25:1, 10:1, 5:1). This procedure resulted in 40 mg of 14 as a bluesolid (0.046 mmol, 90%). R_(f)=0.2 in 9:1 acetonitrile/water. ¹H NMR(600 MHz, MeOD): δ 7.88 (d, J=6.4 Hz, 4H), 7.68 (d, J=7.8 Hz, 4H), 7.54(d, J=7.1 Hz, 4H), 7.22 (s, 4H), 4.32-4.11 (m, 3H), 1.30 (d, J=6.5 Hz,12H). The NMR was taken in the presence of I₂ to keep all of 14 in theneutral oxidation state. The anionic form is paramagnetic which preventsa spectrum form being obtained. Due to this difficulty we havecharacterized the sulfonated Ni bis(dithiolenes) by UV/Vis/NIR, HRMS,and HPLC instead of NMR. UV/Vis/NIR (water): 851 nm (1.2 au), 638 nm(0.4 au), 312 nm (3.1 au), 282 nm (3.3 au), 209 nm (5.1 au), 206 nm (4.7au). HRMS (ESI): calcd. for C₃₆H₃₂O₈N₂NiS₆ ²[M−2H]⁻², 434.9924; found,434.9918.

Compound 11.

4-(2-Oxo-5-(4-sulfophenyl)-1,3-dithiol-4-yl)benzoic acid 9 (20 mg, 0.046mmol, 1 equiv.) was dissolved in dimethylformamide (1 mL, anhydrous). Tothis solution, biotin-(PEG)₃-amine (23 mg, 0.056 mmol, 1.1 equiv.),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl, 15mg, 0.078 mmol, 1.7 equiv.), hydroxybenzotriazole (HOBt, 10 mg, 0.065mmol, 1.4 equiv.), and NEt₃ (15 μL, 0.11 mmol, 2.0 equiv.) were added.The mixture was stirred at room temperature overnight. The followingmorning the mixture was evaporated to dryness and purified by HPLC usingwater/methanol solvent system. Compound 11 eluted at 32 min when agradient of 0 to 100% methanol over 45 min was used. This procedureresulted in pure 11 (17 mg, 0.021 mol, 45%). R_(f)=0.4 in 4:1acetonitrile/methanol. ¹H NMR (500 MHz, CDCl₃): δ 7.71 (d, J=7.9 Hz,4H), 7.31 (t, J=7.6 Hz, 4H), 4.53-4.50 (m, 1H), 4.34-4.32 (m, 1H),3.57-3.52 (m, 8H), 3.49-3.48 (m, 2H), 3.42 (dd, J=10.2, 5.9 Hz, 4H),3.23-3.16 (m, 4H), 3.11-3.07 (m, 1H), 2.89 (dd, J=12.8, 4.8 Hz, 1H),2.69 (d, J=12.8 Hz, 1H), 2.18 (t, J=7.3 Hz, 2H), 1.83-1.81 (m, 2H),1.72-1.58 (m, 6H), 1.41-1.37 (m, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 190.8,176.4, 167.0, 166.0, 147.3, 136.4, 135.9, 134.7, 131.1, 131.0, 130.3,130.0, 129.1, 127.8, 71.6, 71.4, 71.3, 70.4, 70.0, 64., 62.4, 57.0,50.0, 43.5, 41.0, 39.0, 38.2, 36.7, 30.4, 29.9, 29.5, 27.0. HRMS (ESI):cacld. For C₃₆H₄₅O₁₀N₄S₄ ⁻ [M−H]⁻, 821.2024; found, 821.2036.

Ni bis(dithiolene) 15

Ligand precursor 11 (17 mg, 0.021 mmol, 1.0 equiv.) was dissolved in amixture of THF/MeOH (0.2 mL/0.2 mL). Tetramethyl ammonium hydroxidepentahydrate (8 mg, 0.044 mmol, 2.1 equiv.) was added to the solutionwith compound 11. The mixture turned a yellow color. After 30 min,NiCl₂.6H₂O (2.5 mg, 0.011 mmol, 0.50 equiv.) was added and a brownprecipitate formed. This mixture was stirred at room temperatureovernight. The following morning, iodine (2.5 mg, 0.0098 mmol, 0.47equiv.) was added and the mixture turned dark blue in color with most ofthe precipitate returning to solution. After 4 h stirring at roomtemperature, the mixture was evaporated to dryness and purified bysilica gel chromatography eluting with acetonitrile/water (10:1, 5:1,3:1, 2:1). This procedure resulted in 5 mg of 15 as a blue solid (0.0061mmol, 29%). R_(f)=0.7 in 9:1 acetonitrile/water. UV/Vis/NIR (water): 868nm (0.6 au), 349 nm (0.9 au), 315 nm (1.5 au), 276 nm (1.7 au), 201 nm(3.9 au). HRMS (ESI): calcd. for C₇₀H₉₀O₁₈N₈NiS₈ ²⁻ [M−2H]²⁻, 822.1752;found, 822.1749.

7-acetoxy-2,5-norbornadiene (4)

7-tert-Butoxy-2,5-norbornadiene S1 (1.7 g, 10 mmol, 1.0 equiv.) wascombined with acetic anhydride (3.4 mL, 36 mmol, 3.6 equiv.) and aceticacid (16.9 mL) at 0° C. This solution was poured into precooledperchloric acid (2.3 mL, 60%). The yellow reaction mixture was stirredfor 1 min at 0° C. and then poured onto ice water (˜50 mL). Additionalwater was added until no yellow color remained. The aqueous solution wasextracted with dichloromethane (3×50 mL). The organic layers werecombined, dried, decanted, and evaporated to dryness. The crude productwas purified by silica gel chromatography eluting with 25:1hexanes/ether. This procedure resulted in 910 mg pure 4 as a colorlessoil (6.1 mmol, 58%). R_(f)=0.5 in 10:1 hexanes/ethyl acetate. ¹H NMR(600 MHz, CDCl₃): δ 6.63 (s, 2H), 6.50 (s, 2H), 4.50 (s, 1H), 3.53 (s,2H), 1.89 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 171.2, 140.4, 137.9,99.4, 52.5, 21.3. HRMS (EI): calcd. for C₉H₁₀O₂ ⁺ [M]⁺, 150.0681; found,150.0638.

7-acetoxy quadricyclane (1)

7-Acetoxy-2,5-norbornadiene 4 (400 μL, 3.9 mmol, 1.0 equiv.) wasdissolved in hexane (150 mL, degassed) and placed in a quartz roundbottom flask containing a small amount of acetone (˜0.5 mL). The mixturewas irradiated with a 450 W Mercury Arc lamp for 5 h. Throughout theirradiation process, the reaction was kept under a nitrogen atmosphere.Following irradiation, sat AgNO₃ (5 mL) was added and the mixture wasvigorously stirred in the dark for 15 min to complex any remaining 4.The hexane was removed and the aqueous solution was filtered andextracted with hexanes (2×10 mL). The hexanes were combined, dried,decanted, and evaporated to dryness to result in pure 5 as a wet,colorless solid (560 mg, 3.7 mmol, 95%). R_(f)=0.7 in 5:1 hexanes/ethylacetate. ¹H NMR (400 MHz, CDCl₃): δ 5.62 (t, J=1.7 Hz, 1H), 2.11 (s,3H), 1.83-1.80 (m, 2H), 1.62-1.59 (m, 2H), 1.53-1.51 (m, 2H). ¹³C NMR(125 MHz, CDCl₃): δ 171.9, 82.4, 25.8, 21.5, 16.1, 14.8. HRMS (EI):calcd. for C₉H₁₀O₂ ⁺ [M]⁺, 150.0681; found, 150.0638.

7-hydroxy quadricyclane (S2)

7-Acetoxy quadricyclane 1 (325 mg, 2.2 mmol, 1.0 equiv.) was dissolvedether (1.0 mL, anhydrous). This solution was added to lithium aluminumhydride (1.2 mL, 1.2 mmol, 0.55 equiv., 1 M in diethyl ether) precooledto 0° C. The mixture was warmed to room temperature and stirred for 15min, at which point the reaction was quenched with aqueous Rochelle'ssalt (˜5 mL). The mixture was stirred until the aluminum wassufficiently complexed and two layers formed in the flask. The aqueouslayer was extracted with ether (3×20 mL) and the organic layers werecombined, dried with MgSO₄, filtered, and evaporated to dryness. Thisprocedure resulted in ˜90% pure S2 as a volatile, colorless oil (210 mg,1.8 mmol, 80% yield). Note: If this compound is purified by silica gelchromatography and aldehyde byproduct is formed resulting in a less pureproduct than that obtained from the crude reaction. R_(f)=0.2 in 5:1hexanes/ethyl acetate. ¹H NMR (400 MHz, CDCl₃): δ 4.87 (t, J=1.8 Hz,1H), 1.77-1.74 (m, 2H), 1.56-1.53 (m, 2H), 1.38-1.36 (m, 2H). ¹³C NMR(125 MHz, CDCl₃): δ 79.5, 29.0, 15.9, 14.9 HRMS (EI): calcd. for C₇H₈O⁺[M]⁺, 108.0575; found, 108.0574.

p-Nitrophenyl carbonate quadricyclane 16

7-Hydroxy quadricyclane S2 (20 mg, 0.19 mmol, 1.0 equiv.) was dissolvedin dichloromethane (7 mL, anhydrous) and cooled to 0° C. Pyridine (90μL, 1.1 mmol, 5.8 equiv., anhydrous) was added followed by p-nitrophenylchloroformate (87 mg, 0.44 mmol, 2.3 equiv.). The reaction mixture waswarmed to room temperature over 3 h, at which point it was quenched withwater and extracted with dichloromethane (3×15 mL). The organic layerswere combined, dried with MgSO₄, decanted, and evaporated to dryness.The crude product was purified by silica gel chromatography withhexanes/ether (9:1, 4:1, 2:1). This procedure resulted in 27 mg of pure16 as a white solid (0.010 mmol, 52%). R_(f)=0.7 in 7:1 hexanes/ethylacetate. ¹H NMR (600 MHz, CDCl₃): δ 8.28 (d, J=9.0 Hz, 2H), 7.43 (d,J=9.0 Hz, 2H), 5.71 (s, 1H), 1.93-1.91 (m, 2H), 1.70-1.68 (m, 2H),1.65-1.64 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 155.9, 152.9, 145.5,125.5, 122.0, 88.0, 25.8, 16.4, 15.3. HRMS (EI): calcd. for C₁₄H₁₁O₅N⁺[M]⁺, 273.0637; found, 273.0634.

Quadricyclane S4.

Triazole S3 (45 mg, 0.15 mmol, 1.5 equiv.) and diisopropylethylamine(DIPEA) (165 μL, 0.95 mmol, 10 equiv.) were combined in dichloromethane(2 mL, anhydrous) and cooled to 0° C. p-Nitrophenyl carbonatequadricyclane 16 (27 mg, 0.099 mmol, was dissolved in dichloromethane (1mL, anhydrous) and added to the solution containing triazole S3. Thereaction mixture was warmed to room temperature overnight. The followingmorning, the mixture was evaporated to dryness and purified by silicagel chromatography to yield S4 (26 mg, 0.082 mmol, 83%). R_(f)=0.1 in1:1 hexanes/ethyl acetate. ¹H NMR (400 MHz, CDCl₃): δ 7.69 (s, 1H), 5.60(s, 1H), 5.39 (bs, 1H), 5.13 (s, 2H), 4.50 (d, J=6.3 Hz, 2H), 4.26 (q,J=7.1 Hz, 2H), 1.79-1.77 (m, 2H), 1.58 (bs, 2H), 1.49 (bs, 2H), 1.29 (t,J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 166.4, 157.1, 145.8, 123.7,83.1, 62.7, 51.1, 36.6, 26.0, 16.1, 14.8, 14.3. HRMS (ESI): calcd. forC₁₅H₁₉O₄N₄ [M+H]⁺, 319.1401; found, 319.1404.

4,5-diphenyl-1,3-dithiol-2-one (S6)

Diphenylacetylene S3 (2.5 g, 13.9 mmol, 1.0 equiv.) was combined with1,1′-azobis(cyclohexane)carbonitrile (1.5 g, 6.1 mmol, 0.44 equiv.),diisopropyl xanthogen disulfide 7 (4.3 g, 15.9 mmol, 1.1 equiv.) inm-xylene (30 mL, anhydrous). The reaction mixture was heated to refluxovernight. The following day, the reaction mixture was cooled to roomtemperature, evaporated to dryness, and purified by silica gelchromatography with a hexane/toluene solvent system (10:1, 8:1, 6:1).This procedure resulted in 830 mg of S6 (3.1 mmol, 22% yield). R_(f)=0.6in 10:1 hexanes/ethyl acetate. ¹H NMR (400 MHz, CDCl₃): δ 7.35-7.25 (m,10H). ¹³C NMR (100 MHz, CDCl₃): δ 190.7, 131.8, 129.6, 128.93, 128.89,128.8. HRMS (EI): calcd. for C₁₅H₁₀OS₂ ⁺ [M]⁺, 270.0173; found,270.0179.

4,4′-(2-oxo-1,3-dithiole-4,5-diyl)dibenzenesulfonic acid (S7)

4,5-diphenyl-1,3-dithiol-2-one S6 (830 mg, 3.1 mmol, 1.0 equiv.) wasdissolved in sulfuric acid (10 mL, 18 M). Fuming sulfuric acid (150 μL,20% in sulfuric acid) was added and the reaction mixture was heated to90° C. overnight. The following morning the mixture was cooled to 0° C.and neutralized first with NaOH then with NaHCO₃. Once neutral, methanol(100 mL) was added, the solution was filtered, and the filtrateevaporated to dryness. The solid was again dissolved in methanol (100mL), filtered, and the filtrate evaporated to dryness. The residue wasthen dissolved in water (50 mL) and washed with hexane (3×50 mL). Thewater layer was evaporated to dryness and the crude product was purifiedby HPLC on a C18 column with a water/acetonitrile solvent system (0 to30% acetonitrile over 30 min). The product elutes at 10 min. Thisprocedure resulted in 430 mg of pure S7 (1.0 mmol, 32% yield). ¹H NMR(600 MHz, MeOD): δ 7.76 (d, J=8.3 Hz, 4H), 7.33 (d, J=8.4 Hz, 4H). ¹³CNMR (151 MHz, MeOD): δ 191.0, 147.1, 134.6, 130.8, 130.1, 127.8. HRMS(ESI): calcd. for C₁₅H₉O₇S₄ ⁻ [M−H]⁻ 428.9237; found, 428.9238.

Ni bis(dithiolene) 17

4,4′-(2-oxo-1,3-dithiole-4,5-diyl)dibenzenesulfonic acid S7 (33 mg,0.075 mmol, 1.0 equiv.) was dissolved in a mixture of methanol (0.5 mL),THF (0.7 mL), and water (0.75 mL). Tetramethyl ammonium hydroxidepentahydrate (28 mg, 0.15 mmol, 2.1 equiv.) was dissolved in MeOH (0.15mL) and added to the solution with compound S7. The mixture turned ayellow color. After 30 min, NiCl₂.6H₂O (8.5 mg, 0.036 mmol, 0.48 equiv.)was added and stirred for 6 h, at which point iodine (8.5 mg, 0.033mmol, 0.45 equiv.) was added and the blue mixture was stirred overnightat room temperature. The following morning, TLC indicated that somereduced complex may be present and more iodine (5 mg, 0.020 mmol, 0.26equiv.) was added. After stirring for an additional 30 min, the reactionwas evaporated to dryness and purified by silica gel chromatographyeluting with acetonitrile/water (25:1, 9:1). This procedure resulted in28 mg of 17 (0.033 mmol, 43% yield). UV/Vis/NIR: 832 nm (1.1 au), 591 nm(0.3 au), 349 nm (1.0 au), 315 nm (2.0 au), 272 nm (1.7 au). HRMS(ESI):calcd. for C₂₈H₁₆O₁₂NiS₈ ⁴⁻ [M−4H]⁴⁻, 214.4446; found, 214.4447.

DIMAC-Fluorescein (S11).

6,7-dimethoxyazacyclooct-4-yne (DIMAC) (Sletten, E. M.; Bertozzi, C. R.Org. Lett. 2008, 10, 3097-3099; S9, 8.0 mg, 0.030 mmol, 1.0 equiv.) wasdissolved in CH₃CN (1 mL, anhydrous) and cooled to 0° C. DIPEA (10 μL,0.057 mmol, 1.9 equiv.) was added and the mixture was stirred for 10min, at which point pentafluorophenyltrifluoroacetate (15 μL, 0.087mmol, 2.9 equiv.) was added. The reaction was warmed to room temperatureand stirred for 1.5 h. It was then evaporated to dryness and purified bysilica gel chromatography eluting with toluene/ether (7:1, 5:1, 3:1,anhydrous solvents used for chromatography). This procedure resulted inDIMAC-pentafluorophenyl ester (13 mg, 0.030 mmol, quant.). Half of theDIMAC-pentafluorophenyl ester (6.5 mg, 0.015 mmol, 1.0 equiv.) wasdissolved in dimethylformamide (0.5 mL, anhydrous). In a separate flask,fluorescein-piperazine (Hangauer, M. J.; Bertozzi, C. R. Angew. Chem.Int. Ed. 2008, 47, 2394-2397; 11 mg, 0.028 mmol, 1.8 equiv.) wasdissolved in dimethylformamide (0.5 mL, anhydrous) and DIPEA (˜10 μL,0.06 mmol, 4 equiv.). The DIMAC solution was added to thefluorescein-piperazine solution at 0° C. The reaction was warmed to roomtemperature over 5 h, at which point it was evaporated to dryness andpurified first by silica gel chromatography (5:3:1 EtOAc/MeOH/H₂O) thenby HPLC (C18 column, with methanol/water, 40-100% methanol over 25 min,elutes at 15 min). This procedure resulted in pure DIMAC-fluorescein (3mg, 0.005 mmol, 31% yield). R_(f)=0.7 in 5:3:1 ethylacetate/methanol/water. HRMS(ESI): calcd. for C₃₇H₃₇O₈N₃Na [M+Na]⁺,674.2473; found, 674.2478.

Ethyl2-(4-((tert-butoxycarbonylamino)methyl)-1H-1,2,3-triazol-1-yl)acetate(S12)

Ethyl azidoacetate (120 mg, 0.930 mmol, 1.00 equiv.) and N-boc propargylamine (146 mg, 0.942 mmol, 1.02 equiv.) were dissolved in a mixture ofethanol (1.8 mL) and water (1.8 mL). To this solution, CuSO₄ (2 mg, 0.01mmol, 0.1 equiv.) and sodium ascorbate (50 μL of 2M solution in water,0.1 mmol, 1 equiv.) were added. The mixture was stirred overnight atroom temperature. The following morning, the ethanol was removed byevaporation and the product was extracted into ethyl acetate (3×50 mL).The ethyl acetate was dried with MgSO₄, filtered and evaporated todryness. The crude product was purified by silica gel chromatographywith hexanes/ethyl acetate (5:1, 3:1, 1:1, 1:2). This procedure resultedin 178 mg of pure S12 (0.627 mmol, 67%). R_(f)=0.7 in ethyl aceate. ¹HNMR (600 MHz, CDCl₃): δ 7.61 (s, 1H), 5.34 (s, 1H), 5.08 (s, 2H), 4.34(d, J=6.0 Hz, 2H), 4.19 (q, J=7.1 Hz, 2H), 1.37 (s, 9H), 1.23 (t, J=7.1Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 166.3, 155.9, 145.9, 123.4, 79.5,62.3, 50.8, 36.0, 28.3, 14.0. HRMS (ESI): calcd. for C₇H₁₃O₂N₄ [M+H]⁺,185.1039; found, 185.3.

(1-(2-ethoxy-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methanaminium2,2,2-trifluoroacetate (S3)

Boc-protected triazole S12 (178 mg, 0.627 mmol, 1 equiv.) was dissolvedin dichlorometane (8 mL, anhydrous). Trifluoroacetic acid (2 mL) wasadded and this mixture was stirred for 1 h at room temperature, at whichpoint the reaction mixture was evaporated to dryness to yield pure S3(200 mg, 0.67 mmol, quant.). R_(f)=0.7 in 5:3:1 ethylacetate/methanol/water. ¹H NMR (600 MHz, MeOD): δ 8.15 (s, 1H), 5.34 (s,2H), 4.31 (s, 2H), 4.23 (q, J=7.1 Hz, 2H), 1.27 (t, J=7.1 Hz, 3H). ¹³CNMR (151 MHz, MeOD): δ 168.6, 161.3 (q, J=38.3 Hz), 141.5, 127.4, 117.2(q, J=287.3 Hz), 63.6, 52.0, 35.6, 14.4. HRMS (ESI): calcd. forC₁₂H₂₁O₄N₄ [M+H]⁺, 285.1563; found, 285.3.

Example 2: Bioorthogonal Quadricyclane Ligation

To identify a bioorthogonal reaction partner for quadricyclane, avariety of candidates for reactivity were screened at room temperaturein aqueous or polar aprotic solvents. One example of a reagent thatstood out as promising is bis(dithiobenzil)nickel(II) 2, a Nibis(dithiolene) complex that reacted cleanly with 7-acetoxyquadricyclane 1 to yield adduct 3.

The aqueous stability of quadricyclane has been the subject ofconflicting reports, and thus, the stability of 1 was assayed in waterand in the presence of biological nucleophiles. Compound 1 was preparedby the photochemical [2+2] reaction of 4. Compound 1 was found to bestable in phosphate buffered saline (PBS, pH 7.4) with no degradationobserved after more than 2 months at room temperature (FIG. 1). We alsofound Compound 1 to be unreactive with sugars, a variety of oxidants,and free amino acids, most notably cysteine (FIG. 2). Furthermore,quadricyclane was stable in the presence of bovine serum albumin (BSA)and cell culture media under conditions emulating those necessary formetabolic labeling experiments. The limited solubility of Compound 2 inpolar solvents precluded a thorough stability study, but the compoundwas found to be unreactive with a variety of nucleophiles indichloromethane. It is worth noting that the Ni bis(dithiolene) core isconsidered aromatic, no doubt contributing to it general stability.

The stability of the product, norbornene 3, formed by ligation of 1 and2 was studied.

There are reports that quadricyclane's adduct with 2 undergoeslight-induced reversion to norbornadiene, an isomer of quadricyclane,and 2. Accordingly, Compound 3 was exposed to ambient light andmonitored the formation of 2 and 4. The half-life of the reversion was˜35 hours in CDCl₃ (FIGS. 3-4), which would be problematic for manybiological labeling applications.

The mechanism of the photochemical reversion is not well understood, butit is presumed that the reaction could be inhibited by removing Ni fromthe product using a metal chelator. After screening a variety ofoptions, it was found that diethyldithiocarbamate (5) prevented thephotodegradation of 3, as shown below. (FIG. 5).

With these promising results, the synthesis of a Ni bis(dithiolene)reagent that is both water soluble and functionalized with a probe todetect biomolecule labeling was pursued. Compound 15, bearing twosulfonate groups and two biotin moieties, was designed for this purpose.Compound 15 and a model compound, bis(isopropyl amide) 14, fromdithiol-2-one ligand precursors using a previously reported route thatwe felt would be compatible with polar functional groups were prepared.Briefly, alkyne 6 was reacted with xanthogen disulfide 7 in the presenceof the radical initiator 1,1′-azobis(cyclohexanecarbonitrile) to yielddithiocarbonate 8. Treatment of 8 with fuming sulfuric acid installed asingle sulfonate group and also hydrolyzed the methyl ester to produce 9in good yield. Standard amide bond coupling conditions were used toconjugate either isopropyl amine or an amine-functionalized biotinderivative to 9, affording 10 and 11, respectively. These intermediateswere then converted to anionic Ni bis(dithiolene) species 12 and 13 insitu by treatment with tetramethyl ammonium hydroxide and NiCl₂.Immediate subsequent oxidization with 0.5 equivalents of iodine affordedthe desired neutral complexes 14 and 15. Compounds 14 and 15 are solublein PBS at concentrations up to 5 and 10 mM, respectively.

With the solubilities of compounds 14 and 15, reaction kinetics wereassessed with quadricyclane in aqueous/organic solvent mixtures, as wellas to probe in more detail the stability of these Ni bis(dithiolene)complexes in the presence of biomolecules. Complex 14 has a large NIRabsorption band at 850 nm that is not present in the adduct withquadricyclane, allowing for pseudo-first order kinetic measurements byabsorption spectroscopy. The second-order rate constant for the reactionof 14 and 1 in a 1:1 PBS/EtOH mixture was 0.25±0.05 M⁻¹ s⁻¹ at roomtemperature (FIGS. 6-7). This rate constant is comparable to those ofcyclooctyne-azide cycloadditions currently used for biological labelingapplications, and should allow for use of mild reaction conditions (<200μM reagent, <1 h reaction times).

The stability of 14 was also monitored using the NIR absorption band,which is dependent on both the oxidation state of the complex (i.e., 14vs. 12) as well as the connectivity of the dithiolene ligands.Absorption at 850 nm remained essentially unaltered when 14 wasincubated in PBS for 20 h (FIG. 8). As well, exposure to amino acids hadeither no effect or a minimal effect on the absorption intensity (FIG.9). A marked exception was cysteine, which even at 1 molar equivalentcaused an immediate decrease in the 850 nm absorption band's intensityand concomitant appearance of a new absorption at ˜900 nm (FIG. 10).This transformation is consistent with reduction of 14 to 12 andtreatment of 14 with other reducing agents yielded similar results (FIG.11). The neutral complex could be regenerated by addition of potassiumferrocyanide (K₃Fe(CN)₆) as judged by the reappearance of the absorptionband at 850 nm and restoration of reactivity with 1 (FIGS. 12-13).Concerned that the presence of free cysteine residues within proteinswould undergo unwanted redox reactions with 14, we monitored itsintegrity the presence of BSA, which possesses a solvent-exposed reducedcysteine side chain. Incubation with BSA led to reduction of 14 but at amuch slower rate (>1000-fold) than observed with free cysteine.Therefore, protein-mediated reduction should not undermine the muchfaster quadricyclane ligation (FIG. 14). Notably, 14 was stable tooxidized insulin, which contains no free sulfhydryl groups, over a 5hour period (FIG. 15).

The quadricyclane ligation was then subjected to a test ofbioorthogonality: selective protein labeling. The lysine residues on BSAwere modified with quadricyclane p-nitrophenyl carbonate 16 (FIG. 16A).

Preparation of QC-BSA

Bovine serum albumin (100 mg, Sigma) was dissolved in PBS (5 mL).Quadricyclane p-nitrophenyl carbonate 16 (5 mg, 0.02 mmol) was dissolvedin dimethylsulfoxide (DMSO) (300 μL) with a small amount of DMF (60 μL).A portion of the BSA solution (0.5 mL) was combined with thequadricyclane solution (100 μL) and DMSO (200 μL). The mixture instantlyturned yellow indicating release of p-nitrophenol. After 3 hr, theprotein was purified on a NAP-5 column. The column was pre-equilibratedwith PBS (10 mL). A portion of the protein mixture (350 μL) was added tothe column and eluted with PBS (500 μL per fraction). Four fractionswere collected with the second fraction containing the most protein.Protein concentrations were assayed by a NanoDrop2000 (ThermoScientific) and a BioRAD D_(c) assay.

Quadricyclane-modified BSA (QC-BSA) or native BSA were treated with 50μM 15 for various amounts of time and, after quenching with excess 5 and1, assayed the products by Western blot probing with an anti-biotin(α-biotin) antibody conjugated to horse-radish peroxidase (α-biotinHRP).

Western Blot Procedures

The described protein mixtures were quenched with diethyldithiocarbamate5 and quadricyclane 1 (3-15 mM). 4× sodium dodecyl sulfate (SDS)-loadingbuffer (with β-mercaptoethanol (BME)) was added and the protein mixtureswere loaded onto a 12% BisTris gel (BioRAD, Criterion). The gel was runat 200 V in 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Proteinswere transferred to nitrocellulose (0.45 μm, BioRAD) over 90 min at 75V. The nitrocellulose was then treated with Ponceau stain and incubatedin blocking buffer (5% BSA in PBS with 0.1% Tween 20 non-ionicdetergent) for 2 h at rt. Anti-biotin antibody conjugated tohorse-radish peroxidase (α-biotin-HRP, Jackson Labs) was added to theblocking buffer (1:100,000 dilution) and incubated at rt for 1 h. Theblot was washed with PBST (PBS with 0.1% Tween 20, 3×10 min) anddetection was performed by chemiluminescence using Pierce SuperSignalWest Pico Chemiluminescent Substrate.

Compound 15 selectively labeled QC-BSA in a time-dependent manner withvery little background labeling of unmodified BSA, even upon prolongedexposure of the Western blot (FIG. 16B). Similarly, when the reactiontime was held constant (30 min) and the concentration of 15 was varied,dose-dependent labeling was observed, again with minimal nonspecificreactivity (FIG. 16C). As well, pretreatment of QC-BSA withtetrasulfonated Ni bis(dithiolene) 17 quenched the protein-boundquadricyclane moiety, as demonstrated by reduced labeling with 15 (FIG.16D). To determine whether the quadricyclane ligation possessed theheightened selectivity required to label target biomolecules within morecomplex samples, 1.5 μg of QC-BSA (or unmodified BSA) was combined with25 μg of E. coli lysate. This mixture was treated with 50 μM 15 for 30min, then quenched as above and analyzed by Western blot. Selectivelabeling of QC-BSA was observed but the signal was weak (FIG. 17). Thediminished signal could be due to reduction of 15 by a species presentin the lysate. When K₃Fe(CN)₆ (1 mM) was added to the reaction mixture,robust and selective labeling of QC-BSA was observed (FIG. 16E).

New additions to the bioorthogonal reaction compendium are powerful whenthey can be used in conjunction with other bioorthogonal chemistries.Thus, whether the quadricyclane ligation can be performed simultaneouslywith two established bioorthogonal reactions, Cu-free click chemistryand oxime formation, which are already known to be mutually compatiblewas determined. A mixture containing equal amounts of QC-BSA (mw ˜66kDa), azidohomoalanine-containing dihydrofolate reductase (AzDHFR, mw˜23 kDa), and aldehyde-tagged maltose binding protein (CHO-MBP, mw ˜42kDa) was treated with nickel complex 15, an azacyclooctyne conjugated tofluorescein (DIMAC-fluor), and an aminooxy-functionalized FLAG peptide(H₂NO-FLAG) (FIG. 18A). After incubation for 3 hours, the mixture wasseparated into 3 portions and each was analyzed by Western blot probingwith one of the following antibodies (where “α” is “anti”):α-biotin-HRP, α-fluorescein-HRP, or α-FLAG-HRP (FIG. 18B).

Western Blot Procedures

The described protein mixture was quenched with diethyldithiocarbamate 5(9.6 mM), quadricyclane 1 (9.6 mM), 2-azidoethanol (14.5 mM), and excess850 mM tris buffer pH 7.2 until the pH was neutralized. 4×SDS loadingbuffer (with BME) was added and the mixture was separated into threeequal portions and loaded onto a 4-12% BisTris gel (BioRAD, Criterion).The gel was run at 150 V in MES buffer. Proteins were transferred tonitrocellulose (0.45 μm, BioRAD) over 120 min at 50 V. Thenitrocellulose was then treated with Ponceau stain and separated intothree sections. Two sections were incubated in BSA blocking buffer (5%BSA in PBST) and the third was incubated with milk blocking buffer (5%non-fat milk in PBST) for 2 h at rt. One BSA-blocked blot was incubatedwith α-biotin-HRP (1:100,000). The other BSA-blocked blot was incubatedwith α-fluorescein-HRP (1:100,000, Invitrogen) and the milk-blocked blotwas incubated with α-FLAG-HRP (1:100,000, Sigma, M2 monoclonal). Allincubations were performed for 1 h at rt and followed by washing withPBST (3×10 min). Detection was performed by chemiluminescence usingPierce SuperSignal West Pico Chemiluminescent Substrate.

As shown in FIG. 18B, each labeling reagent, including 15, reacted onlywith its complementary bioorthogonal partner. Like the cyclooctyne andaminooxy probes, compound 15 showed no significant labeling of proteinslacking its partner (quadricyclane), nor did it interfere with the otherbioorthogonal reactions. Notably, the conditions of this multiplexedreaction (i.e., pH (4.5), temperature (37° C.) and time (3 h)) weretuned to accommodate the oxime ligation, the most sluggish of the threetransformations. The quadricyclane ligation was quite tolerant of theseconditions.

Controls for the banding patterns seen in FIG. 17B are shown in FIG. 19.In FIG. 19A/B, QC-BSA (8 μg) and 15 (150 μM) were combined 37° C., pH4.5. After 3 h, this mixture was basified with 850 mM tris buffer andquenched with excess 1, 5, and 2-azidoethanol. It was then analyzed byWestern blot probing with an α-biotin-HRP (FIG. 19B). In FIG. 19C/D,CHO-MBP (8 μg) and H₂NO-FLAG (1 mM) were combined at 37° C., pH 4.5.After 3 h, this mixture was basified with 850 mM tris buffer andquenched with excess 1, 5, and 2-azidoethanol. It was then analyzed byWestern blot probing with an α-FLAG-HRP antibody (FIG. 19D). In FIG.19E/F, AzDHFR (8 μg) and DIMAC-fluor (250 μM) were combined at 37° C.,pH 4.5. After 3 h, this mixture was basified with 850 mM tris buffer andquenched with excess 1, 5, and 2-azidoethanol. It was analyzed byWestern blot probing with an α-fluorescein-HRP antibody (FIG. 19F).

Example 3: Toxicity Analysis

Jurkat cells (human T-cell lymphoma) were maintained in RPMI-1640 media(Invitrogen Life Technologies) supplemented with 10% fetal bovine serum(FBS), penicillin (100 units/mL), and streptomycin (0.1 mg/mL) in a 5%CO₂ water-saturated atmosphere. The cells were maintained at densitiesbetween 1×10⁵ and 1.6×10⁶ cells/mL.

FIG. 20 shows cytotoxicity of 17 and the adduct of 1 and 17 in relationto NiCl₂ and Cu(I). Jurkat cells were washed twice with FACS buffer (PBSwith 1% FBS) and placed in a 96-well plate with ˜400,000 cells/well(pellet 2500×g, 3 min, 4° C.). The cells were treated with at 0, 10, 25,50, 100, 250, or 500 μM of 17 (blue diamond), the product of 1 and 17(red square), NiCl₂ (purple triangle), or CuSO₄ in the presence of 1 mMTCEP (green cross) for 1 h. The cells were washed three times byresuspension in fluorescence activated cell sorting (FACS) buffer (200μL) followed by concentration by centrifugation (2500×g, 3 min, 4° C.).Following the third wash, the cells were resuspended in 100 μL of 1×binding buffer containing 5 μL of 7-Amino-actinomycin D (7-AAD) and 5 μLof fluorescein isothiocyanate (FITC)-AnnexinV (buffer and reagents fromBD Pharmingen™). The cells were incubated at room temperature in thedark for 15 min, diluted to 500 μL with binding buffer and analyzed byflow cytometry (FL1 vs. FL3) on a BD Biosciences FACSCalibur flowcytometer equipped with a 488-nm argon laser. Plotted is the percentageof cells that do not stain with either 7-AAD or FITC-Annexin-V. Theerror bars represent the standard deviation of three replicate samples.

FIG. 21 shows representative dot plots for the experiment in FIG. 20.FIG. 21A shows cells treated with no reagent. FIG. 21B shows cellstreated with 50 μM of reagent. FIG. 21C shows cells treated with 500 μMreagent. The percentage of cells in the bottom left quadrant is what isplotted in FIG. 20. MFI=mean fluorescence intensity (arbitrary units).

FIG. 22 shows cytotoxicity of diethyldithiocarbamate (5). Jurkat cellswere washed twice with FACS buffer (PBS with 1% FBS) and placed in a96-well plate with ˜500,000 cells/well (pellet 2500×g, 3 min, 4° C.).The cells were treated with 0, 1.25, 2.5, or 5.0 mM of 5 for 1 h. Thecells were washed three times by resuspension in FACS buffer (200 μL)followed by concentration by centrifugation (2500×g, 3 min, 4° C.).Following the third wash, the cells were resuspended in 100 μL of 1×binding buffer and 7.5 μL of 7-AAD and 5 μL of AnnexinV-PE were added(buffer and reagents from BD Pharmingen™). The cells were incubated atroom temperature in the dark for 15 min, diluted to 500 μL with bindingbuffer and analyzed by flow cytometry (FL2 vs. FL3) on a BD BiosciencesFACSCalibur flow cytometer equipped with a 488-nm argon laser. Plottedis the percentage of cells that do not stain with either 7-AAD orAnnexinV-PE. The error bars represent the standard deviation of threereplicate samples.

FIG. 23 shows representative dot plots for the experiment in FIG. 22.The percentage of cells in the bottom left quadrant is what is plottedin FIG. 22. MFI=mean fluorescence intensity (arbitrary units).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-14. (canceled)
 15. A compound selected from: (a) an azo compound ofthe formula (IIa) or (III):

wherein Ar¹ is an optional aryl or substituted aryl group; R¹ and R² areoptional and are independently selected from hydrogen, alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, alkoxy, substituted alkoxy, aryl, substitutedaryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy,aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester,sulfonyl, sulfonyloxy, and thioalkoxy; and Y¹ and Y² are independentlyselected from hydrogen, halogen, a moiety that comprises a reactivegroup that facilitates covalent attachment of a molecule of interest;and a molecule of interests; wherein at least one of Y¹ and Y² is amoiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest or a molecule of interest; (b) acompound of formula (IV) or formula (V):

wherein R¹ and R² are optional and are independently selected fromhydrogen, alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, substituted alkynylene, alkoxy, substitutedalkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl,aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino,substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, andthioalkoxy; and Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest; wherein at least one of Y¹ and Y² is a moiety that comprises areactive group that facilitates covalent attachment of a molecule ofinterest or a molecule of interest; (c) a ketone compound of formula(VIa) or formula (VIb):

wherein R¹ is optional and is selected from alkylene, substitutedalkylene, alkenylene, substituted alkenylene, alkynylene, substitutedalkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl,sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl,sulfonyloxy, and thioalkoxy; and R², R³, and R⁴ are independentlyselected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl,carboxyl ester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, trihalomethyl, aryl, and substituted aryl; each R⁵is independently selected from hydrogen, alkyl, substituted alkyl,hydroxy, alkoxy, substituted alkoxy, amino, substituted amino,aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, and trihalomethyl;and n is number from one to five; and Y¹ is selected from a moiety thatcomprises a reactive group that facilitates covalent attachment of amolecule of interest; and a molecule of interest; and (d) a compound ofthe formula:

wherein R¹, R², R³, R⁴, and R⁵ are optional linkers; Z¹, Z², Z³, Z⁴, andZ⁵ are independently selected from hydrogen and a biomolecule; whereinat least one of Z¹, Z², Z³, Z⁴, and Z⁵ is a biomolecule.
 16. The azocompound of claim 15, wherein the compound is of the formula:

wherein R¹ is optional and is selected from alkylene, substitutedalkylene, alkenylene, substituted alkenylene, alkynylene, substitutedalkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl,sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl,sulfonyloxy, and thioalkoxy; Y¹ is selected from a moiety that comprisesa reactive group that facilitates covalent attachment of a molecule ofinterest; and a molecule of interest; R² is selected from alkyl,substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino,substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano,halogen, nitro, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, and trihalomethyl; and n is a number from zero to four.
 17. Thecompound of claim 15, wherein the compound is an azo compound of theformula:Y¹—R¹—N═N—R²—Y²  (III), wherein R¹ and R² are optional and areindependently selected from hydrogen, alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino,aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino,amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, andthioalkoxy; and Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest; wherein at least one of Y¹ and Y² is a moiety that comprises areactive group that facilitates covalent attachment of a molecule ofinterest or a molecule of interest.
 18. The compound of claim 15,wherein the compound is an alkynyl compound of the formula:

wherein R¹ and R² are optional and are independently selected fromhydrogen, alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, substituted alkynylene, alkoxy, substitutedalkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl,aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino,substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, andthioalkoxy; and Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest; wherein at least one of Y¹ and Y² is a moiety that comprises areactive group that facilitates covalent attachment of a molecule ofinterest or a molecule of interest.
 19. The compound of claim 15,wherein the compound is an alkenyl compound of the formula:

wherein R¹ and R² are optional and are independently selected fromhydrogen, alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, substituted alkynylene, alkoxy, substitutedalkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl,aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino,substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, andthioalkoxy; and Y¹ and Y² are independently selected from hydrogen;halogen; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; and a molecule ofinterest; wherein at least one of Y¹ and Y² is a moiety that comprises areactive group that facilitates covalent attachment of a molecule ofinterest or a molecule of interest.
 20. The compound of claim 15,wherein the compound is a ketone compound of the formula:

wherein R¹ is optional and is selected from alkylene, substitutedalkylene, alkenylene, substituted alkenylene, alkynylene, substitutedalkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl,sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl,sulfonyloxy, and thioalkoxy; and R², R³, and R⁴ are independentlyselected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl,carboxyl ester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, trihalomethyl, aryl, and substituted aryl; and Y¹is selected from a moiety that comprises a reactive group thatfacilitates covalent attachment of a molecule of interest; and amolecule of interest.
 21. The compound of claim 15, wherein the compoundis a ketone compound of the formula:

wherein R¹ is optional and is selected from alkylene, substitutedalkylene, alkenylene, substituted alkenylene, alkynylene, substitutedalkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl,acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl,sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl,sulfonyloxy, and thioalkoxy; and R² and R³ are independently selectedfrom hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substitutedalkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxylester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, and trihalomethyl; each R⁵ is independentlyselected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl,carboxyl ester, cyano, halogen, nitro, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, and trihalomethyl; and n is number fromone to five; Y¹ is selected from a moiety that comprises a reactivegroup that facilitates covalent attachment of a molecule of interest;and a molecule of interest.
 22. The compound of claim 15, wherein themolecule of interest is selected from a detectable label; a toxin, apeptide, a drug; a member of a specific binding pair, and an epitopetag.
 23. The compound of claim 15, wherein the compound is of theformula:

wherein R¹, R², R³, R⁴, and R⁵ are optional linkers; Z¹, Z², Z³, Z⁴, andZ⁵ are independently selected from hydrogen and a biomolecule; whereinat least one of Z¹, Z², Z³, Z⁴, and Z⁵ is a biomolecule.
 24. Thecompound of claim 23, wherein the biomolecule is selected from aminoacids, peptides, monosaccharides, oligosaccharides, fatty acids, lipids,nucleotides, oligonucleotides, enzyme substrates, enzyme inhibitors,sterols, co-factors, and Co-A derivatives.
 25. The compound of claim 23,wherein the linkers for R¹, R², and R³ are independently selected fromalkylene, substituted alkylene, alkenylene, substituted alkenylene,alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl,substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino,acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino,carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy.
 26. A method forchemoselective modification of a biomolecule comprising a quadricyclane,the method comprising reacting a quadracyclane in aquadricyclane-containing biomolecule with a pi electrophile compoundselected from a metal bis(dithiolene) compound, an azo compound, analkynyl compound, an alkenyl compound and a ketone compound, whereinsaid reacting produces a conjugate between the quadricyclane of thequadracyclane-containing biomolecule and a pi electrophile of thecompound.
 27. A method for synthetically modifying a cellular component,the method comprising: introducing a quadricyclane moiety into acellular component, thereby generating a quadricyclane-modified cellularcomponent; and contacting the cell comprising the quadricyclane-modifiedcellular component with a reaction partner comprising a pi-electrophileselected from a metal bis(dithiolene) compound, an azo compound, analkynyl compound, an alkenyl compound and a ketone compound, saidcontacting being under physiological conditions; wherein said contactingwith said reaction partner results in reaction between the quadricyclanegroup of quadricyclane-modified cellular component and thepi-electrophile of the reaction partner, thereby synthetically andcovalently modifying the cellular component.
 28. The method of claim 27,wherein said contacting is in vitro.
 29. The method of claim 27, whereinsaid contacting is ex vivo.
 30. The method of claim 27, wherein saidcontacting is in vivo.